Compositions and methods related to antibodies to staphylococcal protein a

ABSTRACT

Embodiments concern methods and compositions for treating or preventing a bacterial infection, particularly infection by a  Staphylococcus  bacterium. Aspects include methods and compositions for providing a passive immune response against the bacteria. In certain embodiments, the methods and compositions involve an antibody that binds Staphylococcal protein A (SpA).

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/523,751 filed on Aug. 15, 2011, Ser. No. 61/615,083 filed onMar. 23, 2012, Ser. No. 61/618,417 filed on Mar. 30, 2012, and Ser. No.61/674,135 filed on Jul. 20, 2012, all of which are incorporated hereinby reference in their entirety.

This invention was made with government support under AI52747 andAI92711 from the National Institute of Allergy and Infectious Diseases(NIAID) and 1-U54-AI-057153 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the fields of immunology,microbiology, and pathology. More particularly, it concerns methods andcompositions involving antibodies to bacterial proteins and bacterialpeptides used to elicit such antibodies. The proteins includeStaphylococcal protein A (SpA).

BACKGROUND

The number of both community acquired and hospital acquired infectionshave increased over recent years with the increased use of intravasculardevices. Hospital acquired (nosocomial) infections are a major cause ofmorbidity and mortality, more particularly in the United States, wherethey affect more than 2 million patients annually. The most frequentnosocomial infections are urinary tract infections (33% of theinfections), followed by pneumonia (15.5%), surgical site infections(14.8%) and primary bloodstream infections (13%) (Emorl and Gaynes,1993).

Staphylococcus aureus, Coagulase-negative Staphylococci (mostlyStaphylococcus epidermidis), enterococcus spp., Escherichia coli andPseudomonas aeruginosa are the major nosocomial pathogens. Althoughthese pathogens almost cause the same number of infections, the severityof the disorders they can produce combined with the frequency ofantibiotic resistant isolates balance this ranking towards S. aureus andS. epidermidis as being the most significant nosocomial pathogens.

Staphylococcus can cause a wide variety of diseases in humans and otheranimals through either toxin production or invasion. Staphylococcaltoxins are a common cause of food poisoning, as the bacteria can grow inimproperly-stored food.

Staphylococcus epidermidis is a normal skin commensal, which is also animportant opportunistic pathogen responsible for infections of impairedmedical devices and infections at sites of surgery. Medical devicesinfected by S. epidermidis include cardiac pacemakers, cerebrospinalfluid shunts, continuous ambulatory peritoneal dialysis catheters,orthopedic devices and prosthetic heart valves.

Staphylococcus aureus is the most common cause of nosocomial infectionswith a significant morbidity and mortality. It is the cause of somecases of osteomyelitis, endocarditis, septic arthritis, pneumonia,abscesses and toxic shock syndrome.

S. aureus can survive on dry surfaces, increasing the chance oftransmission. Any S. aureus infection can cause the staphylococcalscalded skin syndrome, a cutaneous reaction to exotoxin absorbed intothe bloodstream. S. aureus can also cause a type of septicemia calledpyaemia that can be life-threatening. Methicillin-resistantStaphylococcus aureus (MRSA) has become a major cause ofhospital-acquired infections.

S. aureus and S. epidermidis infections are typically treated withantibiotics, with penicillin being the drug of choice, but vancomycinbeing used for methicillin resistant isolates. The percentage ofstaphylococcal strains exhibiting wide-spectrum resistance toantibiotics has increased, posing a threat to effective antimicrobialtherapy. In addition, the recent appearance of vancomycin-resistant S.aureus strain has aroused fear that MRSA strains for which no effectivetherapy is available are starting to emerge and spread.

An alternative approach to antibiotics in the treatment ofstaphylococcal infections has been the use of antibodies againststaphylococcal antigens in passive immunotherapy. Examples of thispassive immunotherapy involves administration of polyclonal antisera(WO00/15238, WO00/12132) as well as treatment with monoclonal antibodiesagainst lipoteichoic acid (WO98/57994).

The first generation of vaccines targeted against S. aureus or againstthe exoproteins it produces have met with limited success (Lee, 1996)and there remains a need to develop additional therapeutic compositionsfor treatment of staphylococcus infections.

SUMMARY OF THE INVENTION

Staphylococcus aureus is the most frequent cause of bacteremia andhospital-acquired infection in the United States. An FDA approvedvaccine that prevents staphylococcal disease is currently unavailable.

In certain embodiments there are antibody compositions that inhibit,ameliorate, and/or prevent Staphylococcal infection. In particularembodiments, there is a polypeptide that is capable of binding aStaphylococcal SpA protein. The term polypeptide is understood to meanone or more amino acid or polypeptide chains. For example, the termpolypeptide may refer to a single polypeptide chain comprising a heavyor light chain or a coupled heavy and light chain. The term polypeptidemay also refer to an immunoglobulin (Ig) monomer, comprising fourpolypeptide chains; two heavy and two light chains. The term polypeptidemay also refer to dimeric, trimeric, tetrameric or pentameric Igmolecules.

Moreover, in certain embodiments, this SpA-binding polypeptide isdistinguished from other SpA antibodies because it has properties thatare based on being an antibody or derived from an antibody generatedusing a SpA variant as an antigen—not a SpA wild-type protein. The SpAvariant has 1, 2, 3, 4, 5 or more alterations in 1, 2, 3, 4 and or 5 ofthe A, B, C, D, and/or E domains. Furthermore, as discussed herein,these Spa binding polypeptides are capable of specifically binding suchSpA variants, including but not limited to a KKAA domain variation inall five domains as discussed below.

Certain embodiments are directed to a recombinant peptide comprising 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid segments comprising about,at least or at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 aminoacids in length, including all values and ranges there between, that areat least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to amino acidsegments of Staphylococcal SpA (SEQ ID NO:1). For example, the aminoacid segment(s) from a Staphylococcal SpA may be from a non-toxogenicSpA mutant polypeptide (e.g., SpA_(KKAA)). PCT Publication No. WO2011/005341 and PCT Appln. No. PCT/US11/42845, each incorporated hereinby reference, provide a number of non-toxigenic SpA mutant polypeptidesand methods for using the same. In further aspects, there are antibodiesthat specifically bind one or more of these particular amino acidsegments.

Embodiments also provide for the use of SpA antibodies in methods andcompositions for the treatment of bacterial and/or staphylococcalinfection. In certain embodiments, compositions are used in themanufacture of medicaments for the therapeutic and/or prophylactictreatment of bacterial infections, particularly staphylococcusinfections. Furthermore, in some embodiments there are methods andcompositions that can be used to treat (e.g., limiting staphylococcalabscess formation and/or persistence in a subject) or prevent bacterialinfection.

Certain aspects are directed to methods of reducing Staphylococcusinfection or abscess formation comprising administering to a patienthaving or suspected of having a Staphylococcus infection an effectiveamount of one or more purified polypeptides or proteins thatspecifically bind a Staphylococcal SpA polypeptide. It is contemplatedthat this polypeptide (or protein) may be referred to as an antibody byvirtue of it being a polypeptide or protein with amino acid sequences ofor derived from one or more CDR regions of an antibody. Any embodimentdiscussed herein in the context of an antibody may be implemented withrespect to a polypeptide or protein so long as the polypeptide orprotein has one or more amino acid regions that has at least 60%identity or homology across the entire region of a CDR from an antibodythat is capable of specifically binding a SpA variant lacking specificIg-binding activity The Spa binding polypeptide can be a purifiedpolyclonal antibody, a purified monoclonal antibody, a recombinantpolypeptide, or a fragment thereof. In certain aspects the polypeptideis an antibody that is humanized, which means the nonvariable portion ofthe antibody has been altered in order to simulate the constant regionsfound in human antibodies. Thus, it is contemplated that a humanizedantibody is one that has the CDR sequences of a non-human antibody (orat least amino acid sequences that are derived from such sequences,i.e., are at least 80% identical).

In certain other embodiments, the antibody is a human antibody. In stillfurther aspects the antibody is a recombinant antibody segment. Incertain aspects a monoclonal antibody includes one or more of 5A10, 8E2,3A6, 7E2, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3,4C5, 6B2, 4D5, 2B8 or 1H7 described in Tables 1-2 below and in Table 5,incorporated herein by reference. An antibody or polypeptide can beadministered at a dose of 0.1, 0.5, 1, 5, 10, 50, 100 mg or μg/kg to 5,10, 50, 100, 500 mg or μg/kg. The recombinant antibody segment can beoperatively coupled to a second recombinant antibody segment. In certainaspects the second recombinant antibody segment binds a secondStaphylococcal protein. The method can further comprise administering asecond antibody that binds a second Staphylococcal protein. In certainaspects the method further comprises administering an antibiotic.

Embodiments are directed to monoclonal antibody polypeptides,polypeptides having one or more segments thereof, and polynucleotidesencoding the same. In certain aspects a polypeptide can comprise all orpart of the heavy chain variable region and/or the light chain variableregion of SpA specific antibodies. In a further aspect, a polypeptidecan comprise an amino acid sequence that corresponds to a first, second,and/or third complementary determining regions (CDRs) from the lightvariable chain and/or heavy variable chain of an antibody, e.g., aSpA-specific antibody. Additionally an antibody or binding polypeptidemay have a binding region comprising an amino acid sequence having,having at least, or having at most 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100% identity or homology (substitution with a conservedamino acid) (or any range derivable therein) with 1, 2, 3, 4, 5, or 6CDR sequences discussed herein, including any of SEQ ID NOs: 11-13,21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 96-98, 111-113,116-118, 126-128, 131-133, 16-18, 26-28, 36-38, 46-48, 56-58, 66-68,76-78, 86-88, 101-103, 106-108, 121-123, 136-138, 141-143. In specificembodiments, an antibody having all or part of one or more CDRsdisclosed herein has been humanized in non-CDR regions. In furtherembodiments, the CDR regions disclosed herein may be changed by 1, 2, 3,4, 5, 6, 7 or 8 amino acids per CDR, which may be instead of or inaddition to humanization. In some embodiments, a change may be adeletion or addition of 1, 2, or 3 amino acids, or it may be asubstitution of any amino acid, which may or may not be with an aminoacid that is a conserved an amino acid.

In some embodiments, a SpA binding polypeptide or antibody has one, two,three, four, five, six, or seven CDRs that have 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% identity with a consensussequence identified for that CDR, such as is shown in Tables 7-17. It iscontemplated that in some embodiments, a SpA binding polypeptide orantibody has an amino acid sequence corresponding to CDR1, CDR2, andCDR3 of a light chain variable region and a CDR1, CDR2, and CDR3 of aheavy chain variable region. As discussed herein the amino acid sequencecorresponding to a CDR may have a percent identity or homology to a CDRdiscussed herein. In certain embodiments, the consensus sequence is SEQID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149,SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ IDNO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158 orSEQ ID NO:159. In particular embodiments, the SpA binding polypeptide orantibody has a consensus sequence from a monoclonal antibody for CDR1,CDR2, and/or CDR3 of the light chain variable region. Alternatively oradditionally, the SpA binding polypeptide or antibody has a consensussequence from a monoclonal antibody for CDR1, CDR2, and/or CDR3 of aheavy chain variable region. It is further contemplated that a SpAbinding polypeptide or antibody may have a mix of CDRs based onconsensus sequence(s) and/or sequences with identity or homology to aparticular CDR.

In some embodiments a SpA binding polypeptide or antibody has one ormore consensus sequences with respect to 3F6. In particular embodiments,the SpA binding polypeptide or antibody has a consensus sequence from3F6 for CDR1, CDR2, and/or CDR3 of the light chain variable region.Alternatively or additionally, the SpA binding polypeptide or antibodyhas a consensus sequence from 3F6 for CDR1, CDR2, and/or CDR3 of a heavychain variable region.

In certain embodiments, an SpA antibody or binding polypeptide comprisesan amino acid sequence that is at least 40% identical to one or moreantibody CDR domains from a SpA-binding antibody wherein the polypeptidespecifically binds at least two Spa Ig binding domains A, B, C, D, and Eof a Staphylococcal protein A polypeptide variant that lacksnon-specific Ig-binding activity. Further embodiments of this aspect arecontemplated below.

In yet other embodiments, a purified polypeptide that specifically bindsto a SpA variant polypeptide lacking specific Ig-binding activity andwherein the polypeptide has an association constant of 0.5×10⁹ M⁻¹ orgreater for at least two and up to five Spa IgG binding domainsA_(KKAA), B_(KKAA), C_(KKAA), D_(KKAA) and E_(KKAA) is contemplated.Further embodiments of this embodiment are contemplated below.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 3D11 variable (VDJ) heavy chain aminoacid sequence QSGPELMKPGASVKISCKASGYSFTSYYMHWVKQSHGKSLEWIGYIDPFNGGTSYNQKFKGKATLTVDKSSSTAYMHLSSLTSEDSAVYYCARYGYDGTFYAMDYWGQGTSVTVS S. CDRs areindicated in bold underline. CDRs are regions within antibodies wherethe antibody complements an antigen's shape. Thus, CDRs determine theprotein's affinity and specificity for specific antigens. From amino tocarboxy terminus the CDRs are CDR1, CDR2, and CDR3. In certain aspects,a polypeptide can comprise 1, 2, and/or 3 CDRs from the variable heavychain of MAb 3D11, for example, SEQ ID NO:81, SEQ ID NO:82, and/or SEQID NO:83. In further embodiments, an antibody may have CDRs that have 1,2, and/or 3 amino acid changes (addition of 1 or 2 amino acids,deletions or 1 or 2 amino acids or substitution) with respect to these1, 2, or 3 CDRs. In further embodiments, an antibody may bealternatively or additionally humanized in regions outside the CDR(s)and/or variable region(s). In some aspects, a polypeptide comprisesadditionally or alternatively, an amino acid sequence that is at least60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical orhomologous to the amino acid sequence of the variable region that is nota CDR sequence, i.e., the variable region framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 3D11 variable (VJ) light chain aminoacid sequenceRIVLTQSPAITAASLGQKVTITCSASSSVSYMHWYQQKSGTSPKPWIYEISKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQQWSYPFTFGSGTKLEIK. CDRs are indicated inbold underline. From amino to carboxy terminus the CDRs are CDR1, CDR2,and CDR3. In certain aspects, a polypeptide can comprise 1, 2, and/or 3CDRs from the variable light chain of MAb 3D11, for example, SEQ IDNO:86, SEQ ID NO:87, and/or SEQ ID NO:88. In further embodiments, apolypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In other embodiments, a polypeptide or protein comprises 1, 2, 3, 4, 5,or 6 CDRs from the either or both of the light and heavy variableregions of mAb 3D11, and 1, 2, 3, 4, 5, or 6 CDRs may have 1, 2, and/or3 amino acid changes with respect to these CDRs. In some embodiments,parts or all of the antibody sequence outside the variable region havebeen humanized. A protein may comprise one or more polypeptides. In someaspects, a protein may contain one or two polypeptides similar to aheavy chain polypeptide and/or 1 or 2 polypeptides similar to a lightchain polypeptide. In further embodiments, a polypeptide may be a singlechain antibody or other antibody discussed herein so long as it at least70% sequence identity or homology to 1, 2, 3, 4, 5, or 6 CDRs of mAb3D11.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 3F6 variable (VDJ) heavy chain aminoacid sequence EVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFSISRDDSQNMLSLQMNNLKTEDTAIYYCVTEHYDYDYYVMDYW GQGTSVXSPQ.CDRs are indicated in bold underline. From amino to carboxy terminus theCDRs are CDR1, CDR2, and CDR3. In certain aspects, a polypeptide cancomprise 1, 2, and/or 3 CDRs from the variable heavy chain of MAb 3F6,for example, SEQ ID NO:51, SEQ ID NO:52, and/or SEQ ID NO:53. In furtherembodiments, a polypeptide may have CDRs that have 1, 2, and/or 3 aminoacid changes (addition of 1 or 2 amino acids, deletions or 1 or 2 aminoacids or substitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 3F6 variable (VJ) light chain aminoacid sequenceIVLTQSPASLAVSLGQRATISCRASESVEYSGASLMQWYQHKPGQPPKKLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPSTFGGGTKLEIK. CDRs are indicatedin bold underline. From amino to carboxy terminus the CDRs are CDR1,CDR2, and CDR3. In certain aspects, a polypeptide can comprise 1, 2,and/or 3 CDRs from the variable light chain of MAb 3F6, for example, SEQID NO:56, SEQ ID NO:57, and/or SEQ ID NO:58. In further embodiments, apolypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In other embodiments, a polypeptide or protein comprises 1, 2, 3, 4, 5,or 6 CDRs from the either or both of the light and heavy variableregions of mAb 3F6, and 1, 2, 3, 4, 5, or 6 CDRs may have 1, 2, and/or 3amino acid changes with respect to these CDRs. In some embodiments,parts or all of the antibody sequence outside the variable region havebeen humanized. A protein may comprise one or more polypeptides. In someaspects, a protein may contain one or two polypeptides similar to aheavy chain polypeptide and/or 1 or 2 polypeptides similar to a lightchain polypeptide. In further embodiments, a polypeptide may be a singlechain antibody or other antibody discussed herein so long as it at least70% sequence identity or homology to 1, 2, 3, 4, 5, or 6 CDRs of mAb3F6.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 5A10 variable (VDJ) heavy chain aminoacid sequenceEVKLVESGGGLVKPGGSLKLSCAASGFAFSNYDMSWVRQTPEKRLEWVATISSGGTYPYYPDSVKGRFTISRDNAKNTLYLQLSSLRSEDTALYYCARGGFLITTRDYYAMDYWGQGTSVTVSS. CDRs are indicated in bold underline. From amino to carboxyterminus the CDRs are CDR1, CDR2, and CDR3. In certain aspects, apolypeptide can comprise 1, 2, and/or 3 CDRs from the variable heavychain of MAb 5A10, for example, SEQ ID NO:11, SEQ ID NO:12, and/or SEQID NO:13. In further embodiments, a polypeptide may have CDRs that have1, 2, and/or 3 amino acid changes (addition of 1 or 2 amino acids,deletions or 1 or 2 amino acids or substitution) with respect to these1, 2, or 3 CDRs. In further embodiments, an antibody may bealternatively or additionally humanized in regions outside the CDR(s)and/or variable region(s). In some aspects, a polypeptide comprisesadditionally or alternatively, an amino acid sequence that is at least60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical orhomologous to the amino acid sequence of the variable region that is nota CDR sequence, i.e., the variable region framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 5A10 variable (VJ) light chain aminoacid sequenceTIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGGGTKLEIK. CDRs are indicated inbold underline. From amino to carboxy terminus the CDRs are CDR1, CDR2,and CDR3. In certain aspects, a polypeptide can comprise 1, 2, and/or 3CDRs from the variable light chain of MAb 5A10, for example, SEQ IDNO:16, SEQ ID NO:17, and/or SEQ ID NO:18. In further embodiments, apolypeptide may CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In other embodiments, a polypeptide or protein comprises 1, 2, 3, 4, 5,or 6 CDRs from the either or both of the light and heavy variableregions of mAb 5A10, and 1, 2, 3, 4, 5, or 6 CDRs may have 1, 2, and/or3 amino acid changes with respect to these CDRs. In some embodiments,parts or all of the antibody sequence outside the variable region havebeen humanized. A protein may comprise one or more polypeptides. In someaspects, a protein may contain one or two polypeptides similar to aheavy chain polypeptide and/or 1 or 2 polypeptides similar to a lightchain polypeptide. In further embodiments, a polypeptide may be a singlechain antibody or other antibody discussed herein so long as it at least70% sequence identity or homology to 1, 2, 3, 4, 5, or 6 CDRs of mAb5A10.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 2F2 variable (VDJ) heavy chain aminoacid sequence VKLVESGGDLVKPGGSLKLSCAASRFTFSSYVMSWVRQTPEKRLEWVASIGSGGTTYYPDTVKGRFTISRDNARNILYLQMSSLRSDDTAMYYCTRGRGYGFAWYFDVWGAGTTV TVSS. CDRs areindicated in bold underline. From amino to carboxy terminus the CDRs areCDR1, CDR2, and CDR3. In certain aspects, a polypeptide can comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb 2F2, for example,SEQ ID NO:96, SEQ ID NO:97, and/or SEQ ID NO:98. In further embodiments,a polypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 2F2 variable (VJ) light chain aminoacid sequenceTIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGGGTKLEIK. CDRs are indicated inbold underline. From amino to carboxy terminus the CDRs are CDR1, CDR2,and CDR3. In certain aspects, a polypeptide can comprise 1, 2, and/or 3CDRs from the variable light chain of MAb 2F2, for example, SEQ IDNO:101, SEQ ID NO:102, and/or SEQ ID NO:103. In further embodiments, apolypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In other embodiments, a polypeptide or protein comprises 1, 2, 3, 4, 5,or 6 CDRs from the either or both of the light and heavy variableregions of mAb 2F2, and 1, 2, 3, 4, 5, or 6 CDRs may have 1, 2, and/or 3amino acid changes with respect to these CDRs. In some embodiments,parts or all of the antibody sequence outside the variable region havebeen humanized. A protein may comprise one or more polypeptides. In someaspects, a protein may contain one or two polypeptides similar to aheavy chain polypeptide and/or 1 or 2 polypeptides similar to a lightchain polypeptide. In further embodiments, a polypeptide may be a singlechain antibody or other antibody discussed herein so long as it at least70% sequence identity or homology to 1, 2, 3, 4, 5, or 6 CDRs of mAb2F2.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 4C5 variable (VJ) light chain aminoacid sequenceDIVLTQSPASLAVSLGQRATISCRASESVEYYGASLMQWYQQKSGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPNTFGGGTKLEIK. CDRs are indicatedin bold underline. From amino to carboxy terminus the CDRs are CDR1,CDR2, and CDR3. In certain aspects, a polypeptide can comprise 1, 2,and/or 3 CDRs from the variable light chain of MAb 4C5, for example, SEQID NO:136, SEQ ID NO:137, and/or SEQ ID NO:138. In further embodiments,a polypeptide may CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In other embodiments, a polypeptide or protein comprises 1, 2, 3, 4, 5,or 6 CDRs from the either or both of the light and heavy variableregions of mAb 4C5, and 1, 2, 3, 4, 5, or 6 CDRs may have 1, 2, and/or 3amino acid changes with respect to these CDRs. In some embodiments,parts or all of the antibody sequence outside the variable region havebeen humanized. A protein may comprise one or more polypeptides. In someaspects, a protein may contain one or two polypeptides similar to aheavy chain polypeptide and/or 1 or 2 polypeptides similar to a lightchain polypeptide. In further embodiments, a polypeptide may be a singlechain antibody or other antibody discussed herein so long as it at least70% sequence identity or homology to 1, 2, 3, 4, 5, or 6 CDRs of mAb4C5.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 4D5 variable (VJ) light chain aminoacid sequenceEIVLTQSPAITAASLGQKVTITCSASSSVSYMHWYHQKSGTSPKPWIYETSKLASGVPVRFSGSGSGTSYSLTISSMEAEDAAIYYCQQWSYPFTFGSGTKLEIK. CDRs are indicated inbold underline. From amino to carboxy terminus the CDRs are CDR1, CDR2,and CDR3. In certain aspects, a polypeptide can comprise 1, 2, and/or 3CDRs from the variable light chain of MAb 4D8, for example, SEQ IDNO:141, SEQ ID NO:142, and/or SEQ ID NO:143. In further embodiments, apolypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 5A11 variable (VDJ) heavy chain aminoacid sequence EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATISDGGTYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCARDRDDYDEGPYFDYWG QGTTLTVSS. CDRsare indicated in bold underline. From amino to carboxy terminus the CDRsare CDR1, CDR2, and CDR3. In certain aspects, a polypeptide can comprise1, 2, and/or 3 CDRs from the variable heavy chain of MAb 5A11, forexample, SEQ ID NO:91, SEQ ID NO:92, and/or SEQ ID NO:93. In furtherembodiments, a polypeptide may have CDRs that have 1, 2, and/or 3 aminoacid changes (addition of 1 or 2 amino acids, deletions or 1 or 2 aminoacids or substitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 6B2 variable (VJ) light chain aminoacid sequenceDIVLTQSPASLAVSLGQRATISCRASESVDYSGASLMQWYQHKPGQPPRLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPSTFGGGTKLEIK. CDRs are indicatedin bold underline. From amino to carboxy terminus the CDRs are CDR1,CDR2, and CDR3. In certain aspects, a polypeptide can comprise 1, 2,and/or 3 CDRs from the variable light chain of MAb 6B2, for example, SEQID NO:121, SEQ ID NO:122, and/or SEQ ID NO:123. In further embodiments,a polypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 8E2 variable (VDJ) heavy chain aminoacid sequence KVQLQQSGAGLVKPGASVKLSCKASGYTFTEYSIHWVKQSSGQGLEWIGWFYPGSGYIKYNEKFKDKATLTADKSSSTVYMEFSRLTSEDSAVYFCARHGYGNYVGYAMDYWG QGTSVTVSS. CDRsare indicated in bold underline. From amino to carboxy terminus the CDRsare CDR1, CDR2, and CDR3. In certain aspects, a polypeptide can comprise1, 2, and/or 3 CDRs from the variable heavy chain of MAb 8E2, forexample, SEQ ID NO:21, SEQ ID NO:22, and/or SEQ ID NO:23. In furtherembodiments, a polypeptide may have CDRs that have 1, 2, and/or 3 aminoacid changes (addition of 1 or 2 amino acids, deletions or 1 or 2 aminoacids or substitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 8E2 variable (VJ) light chain aminoacid sequenceDIQMTQSPASLSASVGETVTITCRASEIIYSYLAWYQQKQGKSPQLLVYFAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGIYYCQHHYGTPYTFGGGTKLEIK. CDRs are indicated inbold underline. From amino to carboxy terminus the CDRs are CDR1, CDR2,and CDR3. In certain aspects, a polypeptide can comprise 1, 2, and/or 3CDRs from the variable light chain of MAb 8E2, for example, SEQ IDNO:26, SEQ ID NO:27, and/or SEQ ID NO:28. In further embodiments, apolypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In other embodiments, a polypeptide or protein comprises 1, 2, 3, 4, 5,or 6 CDRs from the either or both of the light and heavy variableregions of mAb 8E2, and 1, 2, 3, 4, 5, or 6 CDRs may have 1, 2, and/or 3amino acid changes with respect to these CDRs. In some embodiments,parts or all of the antibody sequence outside the variable region havebeen humanized. A protein may comprise one or more polypeptides. In someaspects, a protein may contain one or two polypeptides similar to aheavy chain polypeptide and/or 1 or 2 polypeptides similar to a lightchain polypeptide. In further embodiments, a polypeptide may be a singlechain antibody or other antibody discussed herein so long as it at least70% sequence identity or homology to 1, 2, 3, 4, 5, or 6 CDRs of mAb8E2.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 3A6 variable (VDJ) heavy chain aminoacid sequence QIQLVQSGPELKKPGETVKISCKASGYNFTDYSMHWVKQAPGKGLKWVGWINTETAESTYADDFKGRFAFSLETSASTAYLQINSLKDEDTATFFCAHFDCWGQGTTLTVSS. CDRs areindicated in bold underline. From amino to carboxy terminus the CDRs areCDR1, CDR2, and CDR3. In certain aspects, a polypeptide can comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb 3A6, for example,SEQ ID NO:31, SEQ ID NO:32, and/or SEQ ID NO:33. In further embodiments,a polypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 3A6 variable (VJ) light chain aminoacid sequenceDVVMTQISLSLPVTLGDQASISCRASQSLVHSNGNTYLNWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQITYVPWTFGGGTKLEIK. CDRs areindicated in bold underline. From amino to carboxy terminus the CDRs areCDR1, CDR2, and CDR3. In certain aspects, a polypeptide can comprise 1,2, and/or 3 CDRs from the variable light chain of MAb 3A6, for example,SEQ ID NO:36, SEQ ID NO:37, and/or SEQ ID NO:38. In further embodiments,a polypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In other embodiments, a polypeptide or protein comprises 1, 2, 3, 4, 5,or 6 CDRs from the either or both of the light and heavy variableregions of mAb 3A6, and 1, 2, 3, 4, 5, or 6 CDRs may have 1, 2, and/or 3amino acid changes with respect to these CDRs. In some embodiments,parts or all of the antibody sequence outside the variable region havebeen humanized. A protein may comprise one or more polypeptides. In someaspects, a protein may contain one or two polypeptides similar to aheavy chain polypeptide and/or 1 or 2 polypeptides similar to a lightchain polypeptide. In further embodiments, a polypeptide may be a singlechain antibody or other antibody discussed herein so long as it at least70% sequence identity or homology to 1, 2, 3, 4, 5, or 6 CDRs of mAb3A6.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 6D11 variable (VDJ) heavy chain aminoacid sequenceQVQLQQSGAELVRPGTSVKVSCKASGNAFTNYLIEWIKQRPGQGLEWIGVINPGSGITNYNEKFKGKATLTADKSSNTAYMQLSSLSSDDSAVYFCSGSANWFAYWGQGTLVTVSA. CDRs areindicated in bold underline. From amino to carboxy terminus the CDRs areCDR1, CDR2, and CDR3. In certain aspects, a polypeptide can comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb 6D11, for example,SEQ ID NO:71, SEQ ID NO:72, and/or SEQ ID NO:73. In further embodiments,a polypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 6D11 variable (VJ) light chain aminoacid sequenceHCAHPSPASLAVSLGQRASISCRASESVEYSGASLMQWYQHKPGQPPKLLIYAASNVESGVPVRFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPSTFGGGTKLEIK CDRs are indicatedin bold underline. From amino to carboxy terminus the CDRs are CDR1,CDR2, and CDR3. In certain aspects, a polypeptide can comprise 1, 2,and/or 3 CDRs from the variable light chain of MAb 6D11, for example,SEQ ID NO:76, SEQ ID NO:77, and/or SEQ ID NO:78. In further embodiments,a polypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In other embodiments, a polypeptide or protein comprises 1, 2, 3, 4, 5,or 6 CDRs from the either or both of the light and heavy variableregions of mAb 6D11, and 1, 2, 3, 4, 5, or 6 CDRs may have 1, 2, and/or3 amino acid changes with respect to these CDRs. In some embodiments,parts or all of the antibody sequence outside the variable region havebeen humanized. A protein may comprise one or more polypeptides. In someaspects, a protein may contain one or two polypeptides similar to aheavy chain polypeptide and/or 1 or 2 polypeptides similar to a lightchain polypeptide. In further embodiments, a polypeptide may be a singlechain antibody or other antibody discussed herein so long as it at least70% sequence identity or homology to 1, 2, 3, 4, 5, or 6 CDRs of mAb6D11.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 8D4 variable (VDJ) heavy chain aminoacid sequence QVQLQQSGAELVRPGASVKISCKAFGSTFTNHHINWVKQRPGQGLDWIGYLNPYNDYTNYNQKFKGKATLTIDKSSSTAYLELSSLTSEDSAVYYCATITFDSQXQ. CDRs are indicated inbold underline. From amino to carboxy terminus the CDRs are CDR1, CDR2,and CDR3. In certain aspects, a polypeptide can comprise 1, 2, and/or 3CDRs from the variable heavy chain of MAb 8D4, for example, SEQ IDNO:111, SEQ ID NO:112, and/or SEQ ID NO:113. In further embodiments, apolypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 1F10 variable (VDJ) heavy chain aminoacid sequenceKELISSKSEEEKWPGTSVKVSCKASGNAFTNYLIEWIKQRPGQGLEWIGVINPGSGITNYNEKFKGKATLTADKSSNTAYMQLSSLSSDDSAVYFCSGSANWFAYWGQGTLVTVSA. CDRs areindicated in bold underline. From amino to carboxy terminus the CDRs areCDR1, CDR2, and CDR3. In certain aspects, a polypeptide can comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb 1F10, for example,SEQ ID NO:61, SEQ ID NO:62, and/or SEQ ID NO:63. In further embodiments,a polypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 1F10 variable (VJ) light chain aminoacid sequence. CDRs are indicated in bold underlineCSPSPASLAVSLGQRATISCRASESVEYSGASLMQWYQHKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPSTFGGGTKLEIK. From amino to carboxyterminus the CDRs are CDR1, CDR2, and CDR3. In certain aspects, apolypeptide can comprise 1, 2, and/or 3 CDRs from the variable lightchain of MAb 1F10, for example, SEQ ID NO:66, SEQ ID NO:67, and/or SEQID NO:68. In further embodiments, a polypeptide may have CDRs that have1, 2, and/or 3 amino acid changes (addition of 1 or 2 amino acids,deletions or 1 or 2 amino acids or substitution) with respect to these1, 2, or 3 CDRs. In further embodiments, an antibody may bealternatively or additionally humanized in regions outside the CDR(s)and/or variable region(s). In some aspects, a polypeptide comprisesadditionally or alternatively, an amino acid sequence that is at least60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical orhomologous to the amino acid sequence of the variable region that is nota CDR sequence, i.e., the variable region framework.

In other embodiments, a polypeptide or protein comprises 1, 2, 3, 4, 5,or 6 CDRs from the either or both of the light and heavy variableregions of mAb 11′10, and 1, 2, 3, 4, 5, or 6 CDRs may have 1, 2, and/or3 amino acid changes with respect to these CDRs. In some embodiments,parts or all of the antibody sequence outside the variable region havebeen humanized. A protein may comprise one or more polypeptides. In someaspects, a protein may contain one or two polypeptides similar to aheavy chain polypeptide and/or 1 or 2 polypeptides similar to a lightchain polypeptide. In further embodiments, a polypeptide may be a singlechain antibody or other antibody discussed herein so long as it at least70% sequence identity or homology to 1, 2, 3, 4, 5, or 6 CDRs of mAb1F10.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 4C1 variable (VJ) light chain aminoacid sequence. CDRs are indicated in bold underlineVLTQSPASLAVSLGQRATISCRASESVEYSGASLMQWYQHKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPSTFGGGTKLEIK. From amino tocarboxy terminus the CDRs are CDR1, CDR2, and CDR3. In certain aspects,a polypeptide can comprise 1, 2, and/or 3 CDRs from the variable lightchain of MAb 4C1, for example, SEQ ID NO:106, SEQ ID NO:107, and/or SEQID NO:108. In further embodiments, a polypeptide may have CDRs that have1, 2, and/or 3 amino acid changes (addition of 1 or 2 amino acids,deletions or 1 or 2 amino acids or substitution) with respect to these1, 2, or 3 CDRs. In further embodiments, an antibody may bealternatively or additionally humanized in regions outside the CDR(s)and/or variable region(s). In some aspects, a polypeptide comprisesadditionally or alternatively, an amino acid sequence that is at least60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical orhomologous to the amino acid sequence of the variable region that is nota CDR sequence, i.e., the variable region framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 2B8 variable (VJ) light chain aminoacid sequenceFFGVSLGQRASISCRASESVEYSGASLIQWYQHKPGQPPKLLIYAASNVESGVPVRFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPSTFGGGTKLEIK. CDRs are indicated in boldunderline. From amino to carboxy terminus the CDRs are CDR1, CDR2, andCDR3. In certain aspects, a polypeptide can comprise 1, 2, and/or 3 CDRsfrom the variable light chain of MAb 2B8, for example, SEQ ID NO:126,SEQ ID NO:127, and/or SEQ ID NO:128. In further embodiments, apolypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 2C3 variable (VDJ) heavy chain aminoacid sequenceEVKLVESGGGLVKPGGSLKLSCAASGFTFSNYDMSWVRQTPEKRLEWVATISSGGTYPYYPDSVKGRFTISRDNAENTLYLQLSSLRSEDTALYYCARGGFLITTRDYYAMDYWGQGTSVTVSS. CDRs are indicated in bold underline. From amino to carboxyterminus the CDRs are CDR1, CDR2, and CDR3. In certain aspects, apolypeptide can comprise 1, 2, and/or 3 CDRs from the variable heavychain of MAb 2C3, for example, SEQ ID NO:131, SEQ ID NO:132, and/or SEQID NO:133. In further embodiments, a polypeptide may have CDRs that have1, 2, and/or 3 amino acid changes (addition of 1 or 2 amino acids,deletions or 1 or 2 amino acids or substitution) with respect to these1, 2, or 3 CDRs. In further embodiments, an antibody may bealternatively or additionally humanized in regions outside the CDR(s)and/or variable region(s). In some aspects, a polypeptide comprisesadditionally or alternatively, an amino acid sequence that is at least60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical orhomologous to the amino acid sequence of the variable region that is nota CDR sequence, i.e., the variable region framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 7E2 variable (VDJ) heavy chain aminoacid sequence QIQLVQSGPELKKPGETVKISCKASGYTFTDYSVHWVKQAPGKGLKWMAWINTATGEPTFADDFKGRFAFSLETSARTAYLQINNLKNEDTATYFCAPQLTGPFAYWGHGTLVTV SA. CDRs areindicated in bold underline. CDRs are regions within antibodies wherethe antibody complements an antigen's shape. Thus, CDRs determine theprotein's affinity and specificity for specific antigens. From amino tocarboxy terminus the CDRs are CDR1, CDR2, and CDR3. In certain aspects,a polypeptide can comprise 1, 2, and/or 3 CDRs from the variable heavychain of MAb 7E2, for example, SEQ ID NO:41, SEQ ID NO:42, and/or SEQ IDNO:43. In further embodiments, a polypeptide may have CDRs that have 1,2, and/or 3 amino acid changes (addition of 1 or 2 amino acids,deletions or 1 or 2 amino acids or substitution) with respect to these1, 2, or 3 CDRs. In further embodiments, an antibody may bealternatively or additionally humanized in regions outside the CDR(s)and/or variable region(s). In some aspects, a polypeptide comprisesadditionally or alternatively, an amino acid sequence that is at least60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical orhomologous to the amino acid sequence of the variable region that is nota CDR sequence, i.e., the variable region framework.

In certain aspects, a polypeptide comprises all or part of an amino acidsequence corresponding to the MAb 7E2 variable (VJ) light chain aminoacid sequence DIQMTQSPASLSASVGETVTITCRASENIHNYLAWYQQKQGKSPQLLVYNAKTLTDGVPSRFSGSGSGTQFSLKINSLQAGDFGSYYCQHSWSIPYTFGGGTRLQIRR. CDRs are indicatedin bold underline. From amino to carboxy terminus the CDRs are CDR1,CDR2, and CDR3. In certain aspects, a polypeptide can comprise 1, 2,and/or 3 CDRs from the variable light chain of MAb 7E2, for example, SEQID NO:46, SEQ ID NO:47, and/or SEQ ID NO:48. In further embodiments, apolypeptide may have CDRs that have 1, 2, and/or 3 amino acid changes(addition of 1 or 2 amino acids, deletions or 1 or 2 amino acids orsubstitution) with respect to these 1, 2, or 3 CDRs. In furtherembodiments, an antibody may be alternatively or additionally humanizedin regions outside the CDR(s) and/or variable region(s). In someaspects, a polypeptide comprises additionally or alternatively, an aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or 100% identical or homologous to the amino acid sequence ofthe variable region that is not a CDR sequence, i.e., the variableregion framework.

In other embodiments, a polypeptide or protein comprises 1, 2, 3, 4, 5,or 6 CDRs from the either or both of the light and heavy variableregions of mAb 7E2, and 1, 2, 3, 4, 5, or 6 CDRs may have 1, 2, and/or 3amino acid changes with respect to these CDRs. In some embodiments,parts or all of the antibody sequence outside the variable region havebeen humanized. A protein may comprise one or more polypeptides. In someaspects, a protein may contain one or two polypeptides similar to aheavy chain polypeptide and/or 1 or 2 polypeptides similar to a lightchain polypeptide. In further embodiments, a polypeptide may be a singlechain antibody or other antibody discussed herein so long as it at least70% sequence identity or homology to 1, 2, 3, 4, 5, or 6 CDRs of mAb7E2.

In still further aspects, a polypeptide of the embodiments comprises oneor more amino acid segments of the any of the amino acid sequencesdisclosed herein. For example, a polypeptide can comprise 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more amino acid segments comprising about, at least orat most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199 or 200 amino acids in length,including all values and ranges there between, that are at least 80, 85,90, 95, 96, 97, 98, 99, or 100% identical to any of the amino acidsequences disclosed herein. In certain aspects the amino segment(s) areselected from one of the amino acid sequences of a SpA-binding antibodyas provided in Table 5.

In still further aspects, a polypeptide of the embodiments comprises anamino acid segment of the any of the amino acid sequences disclosedherein, wherein the segment begins at amino acid position 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, or 200 in any sequence provided herein andends at amino acid position 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200in the same provided sequence. In certain aspects the amino segment(s),or portions thereof, are selected from one of the amino acid sequencesof a SpA-binding antibody as provided in Table 5.

In yet further aspects, a polypeptide of the embodiments comprises anamino acid segment that is at least 80, 85, 90, 95, 96, 97, 98, 99, or100% identical (or any range derivable therein) to a V, VJ, VDJ, D, DJ,J or CDR domain of a SpA-binding antibody (as provided in Table 5). Forexample, a polypeptide may comprise 1, 2 or 3 amino acid segment thatare at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical (or anyrange derivable therein) to CDRs 1, 2, and/or 3 a SpA-binding antibodyas provided in Table 5.

In further aspects, a nucleic acid molecule of the embodiments comprisesone or more nucleic acid segments of the any of the nucleic acidsequences disclosed herein. For example, a nucleic acid molecule cancomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid segmentscomprising about, at least or at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 ormore nucleotides in length, including all values and ranges therebetween, that are at least 80, 85, 90, 95, 96, 97, 98, 99, or 100%identical (or any range derivable therein) to any of the nucleic acidsequences disclosed herein. In certain aspects, the nucleic acidsegment(s) are selected from one of the nucleic acid sequences encodingportions of SpA-binding antibodies as provided in Table 5.

In yet further aspects, a nucleic acid molecule of the embodimentscomprises a nucleic acid segment that is at least 80, 85, 90, 95, 96,97, 98, 99, or 100% identical (or any range derivable therein) to asequence encoding a V, VJ, VDJ, D, DJ, J or CDR domain of a SpA-bindingantibody as provided in Table 5. For example, a nucleic acid moleculemay comprise 1, 2 or 3 nucleic acid acid segments that are at least 80,85, 90, 95, 96, 97, 98, 99, or 100% identical (or any range derivabletherein) to sequences encoding CDRs 1, 2, and/or 3 a SpA-bindingantibody as provided in Table 5.

In still further aspects, some embodiments provide a hybridoma cell linethat produces a monoclonal antibody. In certain embodiments thehybridoma cell line is a line that produces the 5A10, 8E2, 3A6, 7E2,3F6, 1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2,4D5, 2B8 or 1H7 monoclonal antibody, one or more of which may bedeposited. In a further aspect, 1, 2, and/or 3 CDRs from the lightand/or heavy chain variable region of a MAb can be comprised in ahumanized antibody or variant thereof. In other embodiments, abi-specific antibody is contemplated in which a binding polypeptide iscapable of binding at least two different antigens.

Certain aspects are directed to methods of treating a subject having orsuspected of having a Staphylococcus infection comprising administeringto a patient having or suspected of having a Staphylococcus infection aneffective amount of a purified antibody or binding polypeptide thatspecifically binds a Staphylococcal protein A.

In a further aspect methods are directed to treating a subject at riskof a Staphylococcus infection comprising administering to a patient atrisk of a Staphylococcus infection an effective amount of an antibody orbinding polypeptide that binds a Staphylococcal protein A polypeptideprior to infection with Staphylococcus.

Antibodies or binding polypeptides that are contemplated for use inthese embodiments include those that are able to reduce bacterial load,increase survival, reduce bacterial abscess, confer protective immunity,reduce the number of days on antibiotic, reduce the risk of sepsis orsepticemia, reduce the risk of shock, or provide some other protectiveeffect.

Certain embodiments are directed to a antibody or binding polypeptidecomposition comprising an isolated and/or recombinant antibody orpolypeptide that specifically binds a peptide segment as describedabove. In certain aspects the antibody or polypeptide has a sequencethat is, is at least, or is at most 80, 85, 90, 95, 96, 97, 98, 99, or100% identical (or any range derivable therein) to all or part of anymonoclonal antibody provided herein. In still further aspects theisolated and/or recombinant antibody or polypeptide has, has at least,or has at most 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100 or more contiguous amino acids from any of thesequences provided herein or a combination of such sequences.

In additional embodiments, there are pharmaceutical compositionscomprising one or more polypeptides or antibodies or antibody fragmentsthat are discussed herein. Such a composition may or may not containadditional active ingredients.

In certain embodiments there is a pharmaceutical composition consistingessentially of a polypeptide comprising one or more antibody fragmentsdiscussed herein. It is contemplated that the composition may containnon-active ingredients.

Certain aspects are directed to nucleic acid molecules encoding a heavychain variable regions and/or light chain variable regions of anantibody that specifically binds SpA or a non-toxigenic SpA variant.

Other aspects are directed to pharmaceutical compositions comprising aneffective anti-bacterial amount of an antibody that specifically bindsto a peptide described above and a pharmaceutically acceptable carrier.

The term “providing” is used according to its ordinary meaning toindicate “to supply or furnish for use.” In some embodiments, theprotein is provided directly by administering a composition comprisingantibodies or fragments thereof that are described herein.

The subject typically will have (e.g., diagnosed with a persistentstaphylococcal infection), will be suspected of having, or will be atrisk of developing a staphylococcal infection. Compositions include SpAbinding polypeptides in amounts effective to achieve the intendedpurpose—treatment or protection of Staphylococcal infection. The term“binding polypeptide” refers to a polypeptide that specifically binds toa target molecule, such as the binding of an antibody to an antigen.Binding polypeptides may but need not be derived from immunoglobulingenes or fragments of immunoglobulin genes. More specifically, aneffective amount means an amount of active ingredients necessary toprovide resistance to, amelioration of or mitigation of infection. Inmore specific aspects, an effective amount prevents, alleviates orameliorates symptoms of disease or infection, or prolongs the survivalof the subject being treated. Determination of the effective amount iswell within the capability of those skilled in the art, especially inlight of the detailed disclosure provided herein. For any preparationused in the methods described herein, an effective amount or dose can beestimated initially from in vitro, cell culture, and/or animal modelassays. For example, a dose can be formulated in animal models toachieve a desired response. Such information can be used to moreaccurately determine useful doses in humans.

Compositions can comprise an antibody or a cell that binds SpA. Anantibody can be an antibody fragment, a humanized antibody, a monoclonalantibody, a single chain antibody or the like. In certain aspects, theSpA antibody is elicited by providing a SpA peptide or antigen orepitope that results in the production of an antibody that binds SpA inthe subject. The SpA antibody is typically formulated in apharmaceutically acceptable composition. The SpA antibody compositioncan further comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, or 19 for more staphylococcal antigens orimmunogenic fragments thereof. Staphylococcal antigens include, but arenot limited to all or a segment of Eap, Ebh, Emp, EsaB, EsaC, EsxA,EsxB, IsdA, IsdB, SdrC, SdrD, SdrE, ClfA, ClfB, Coa, Hla (e.g., H35mutants), IsdC, SasF, vWbp, SpA and variants thereof (See U.S.Provisional Application Ser. Nos. 61/166,432, filed Apr. 3, 2009;61/170,779, filed Apr. 20, 2009; and 61/103,196, filed Oct. 6, 2009;each of which is incorporated herein by reference in their entirety), 52kDa vitronectin binding protein (WO 01/60852), Aaa (GenBank CAC80837),Aap (GenBank accession AJ249487), Ant (GenBank accession NP_(—)372518),autolysin glucosaminidase, autolysin amidase, Cna, collagen bindingprotein (U.S. Pat. No. 6,288,214), EFB (FIB), Elastin binding protein(EbpS), EPB, FbpA, fibrinogen binding protein (U.S. Pat. No. 6,008,341),Fibronectin binding protein (U.S. Pat. No. 5,840,846), FnbA, FnbB, GehD(US 2002/0169288), HarA, HBP, Immunodominant ABC transporter, IsaA/PisA,laminin receptor, Lipase GehD, MAP, Mg2+ transporter, MHC II analogue(U.S. Pat. No. 5,648,240), MRPII, Npase, RNA III activating protein(RAP), SasA, SasB, SasC, SasD, SasK, SBI, SdrF (WO 00/12689), SdrG/Fig(WO 00/12689), SdrH (WO 00/12689), SEA exotoxins (WO 00/02523), SEBexotoxins (WO 00/02523), SitC and Ni ABC transporter, SitC/MntC/salivabinding protein (U.S. Pat. No. 5,801,234), SsaA, SSP-1, SSP-2, and/orVitronectin binding protein (see PCT publications WO2007/113222,WO2007/113223, WO2006/032472, WO2006/032475, WO2006/032500, each ofwhich is incorporated herein by reference in their entirety). Thestaphylococcal antigen, or immunogenic fragment or segment can beadministered concurrently with the SpA antibody. The staphylococcalantigen or immunogenic fragment and the SpA antibody can be administeredin the same or different composition and at the same or different times.

The SpA antibody composition can further comprise antibodies, antibodyfragments or antibody subfragments to at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of more staphylococcalantigens or immunogenic fragments thereof. Staphylococcal antigens towhich such antibodies, antibody fragments of antibody subfragments aredirected include, but are not limited to all or a segment of Eap, Ebh,Emp, EsaB, EsaC, EsxA, EsxB, IsdA, IsdB, SdrC, SdrD, SdrE, ClfA, ClfB,Coa, Hla (e.g., H35 mutants), IsdC, SasF, vWbp, SpA and variants thereof(See U.S. Provisional Application Ser. Nos. 61/166,432, filed Apr. 3,2009; 61/170,779, filed Apr. 20, 2009; and 61/103,196, filed Oct. 6,2009; each of which is incorporated herein by reference in theirentirety), 52 kDa vitronectin binding protein (WO 01/60852), Aaa(GenBank CAC80837), Aap (GenBank accession AJ249487), Ant (GenBankaccession NP_(—)372518), autolysin glucosaminidase, autolysin amidase,Cna, collagen binding protein (U.S. Pat. No. 6,288,214), EFB (FIB),Elastin binding protein (EbpS), EPB, FbpA, fibrinogen binding protein(U.S. Pat. No. 6,008,341), Fibronectin binding protein (U.S. Pat. No.5,840,846), FnbA, FnbB, GehD (US 2002/0169288), HarA, HBP,Immunodominant ABC transporter, IsaA/PisA, laminin receptor, LipaseGehD, MAP, Mg2+ transporter, MHC II analogue (U.S. Pat. No. 5,648,240),MRPII, Npase, RNA III activating protein (RAP), SasA, SasB, SasC, SasD,SasK, SBI, SdrF (WO 00/12689), SdrG/Fig (WO 00/12689), SdrH (WO00/12689), SEA exotoxins (WO 00/02523), SEB exotoxins (WO 00/02523),SitC and Ni ABC transporter, SitC/MntC/saliva binding protein (U.S. Pat.No. 5,801,234), SsaA, SSP-1, SSP-2, and/or Vitronectin binding protein(see PCT publications WO2007/113222, WO2007/113223, WO2006/032472,WO2006/032475, WO2006/032500, each of which is incorporated herein byreference in their entirety). The antibodies, antibody fragments orantibody subfragments to other staphylococcal antigens or immunogenicfragments thereof can be administered concurrently with the SpAantibody. The antibodies, antibody fragments or antibody subfragments toother staphylococcal antigens or immunogenic fragments thereof can beadministered in the same or different composition to the SpA antibodyand at the same or different times.

As used herein, the term “modulate” or “modulation” encompasses themeanings of the words “inhibit.” “Modulation” of activity is a decreasein activity. As used herein, the term “modulator” refers to compoundsthat effect the function of a Staphylococcal bacteria, includingpotentiation, inhibition, down-regulation, or suppression of a protein,nucleic acid, gene, organism or the like.

Embodiments include compositions that contain or do not contain abacterium. A composition may or may not include an attenuated or viableor intact staphylococcal bacterium. In certain aspects, the compositioncomprises a bacterium that is not a Staphylococci bacterium or does notcontain Staphylococci bacteria. In certain embodiments a bacterialcomposition comprises an isolated or recombinantly expressed SpAantibody or a nucleic acid encoding the same. In still further aspects,the SpA antibody is multimerized, e.g., a dimer, a trimer, a tertramer,etc.

In certain aspects, a peptide or an antigen or an epitope can bepresented as multimers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptidesegments or peptide mimetics.

The term “isolated” can refer to a nucleic acid or polypeptide that issubstantially free of cellular material, bacterial material, viralmaterial, or culture medium (when produced by recombinant DNAtechniques) of their source of origin, or chemical precursors or otherchemicals (when chemically synthesized). Moreover, an isolated compoundrefers to one that can be administered to a subject as an isolatedcompound; in other words, the compound may not simply be considered“isolated” if it is adhered to a column or embedded in an agarose gel.Moreover, an “isolated nucleic acid fragment” or “isolated peptide” is anucleic acid or protein fragment that is not naturally occurring as afragment and/or is not typically in the functional state.

Compositions such as antibodies, peptides, antigens, or immunogens maybe conjugated or linked covalently or noncovalently to other moietiessuch as adjuvants, proteins, peptides, supports, fluorescence moieties,or labels. The term “conjugate” or “immunoconjugate” is broadly used todefine the operative association of one moiety with another agent and isnot intended to refer solely to any type of operative association, andis particularly not limited to chemical “conjugation.” Recombinantfusion proteins are particularly contemplated.

The term “SpA antibody” refers to polypeptides that bind SpA proteinsfrom staphylococcus bacteria.

In further aspects a composition may be administered more than one timeto the subject, and may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20 or more times. The administration of the compositions include,but is not limited to oral, parenteral, subcutaneous and intravenousadministration, or various combinations thereof, including inhalation oraspiration.

Compositions are typically administered to human subjects, butadministration to other animals that are capable of providing atherapeutic benefit against a staphylococcus bacterium are contemplated,particularly cattle, horses, goats, sheep and other domestic animals,i.e., mammals. In further aspects the staphylococcus bacterium is aStaphylococcus aureus. In still further aspects, the methods andcompositions may be used to prevent, ameliorate, reduce, or treatinfection of tissues or glands, e.g., mammary glands, particularlymastitis and other infections. Other methods include, but are notlimited to prophylactically reducing bacterial burden in a subject notexhibiting signs of infection, particularly those subjects suspected ofor at risk of being colonized by a target bacteria, e.g., patients thatare or will be at risk or susceptible to infection during a hospitalstay, treatment, and/or recovery.

Still further embodiments include methods for providing a subject aprotective or therapeutic composition against a staphylococcus bacteriumcomprising administering to the subject an effective amount of acomposition including (i) a SpA antibody; or, (ii) a nucleic acidmolecule encoding the same, or (iii) administering an SpA antibody withany combination or permutation of bacterial proteins described herein.

The embodiments in the Example section are understood to be embodimentsthat are applicable to all aspects of the invention, includingcompositions and methods.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” It is also contemplatedthat anything listed using the term “or” may also be specificallyexcluded.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention as well as others which will become clear areattained and can be understood in detail, more particular descriptionsand certain embodiments of the invention briefly summarized above areillustrated in the appended drawings. These drawings form a part of thespecification. It is to be noted, however, that the appended drawingsillustrate certain embodiments of the invention and therefore are not tobe considered limiting in their scope.

FIG. 1: SpA_(KKAA)-specific monoclonal antibodies (mAbs) protect miceagainst MRSA infection. Cohorts of animals (n=10) were immunized byintraperitoneal injection with either isotype control (IgG_(2a)) orSpA_(KKAA)-mAb (3F6) at 20 mg·kg⁻¹. After 24 hours post immunization,animals were challenged with 5×10⁶ CFU of S. aureus MW2. (A) At 15 dayspost challenge, animals were euthanized to enumerate the staphylococcalload in kidneys. (B) Serum samples of mice infected for 15 days wereanalyzed for antibodies against the staphylococcal antigen matrix (ClfA,Clumping Factor A; ClfB, Clumping Factor B; FnBPA, Fibronectin BindingProtein A; FnBPB, Fibronectin Binding Protein B; IsdA, Iron surfacedeterminant A; IsdB, Iron surface determinant B; SdrD, Serine-Asparticacid repeat protein D; SpAKKAA, Non-toxigenic staphylococcal protein A;Coa, Coagulase; EsxA, Ess [ESAT-6 (Early Secreted Antigen Target 6 kDa)]secretion system] extracellular A; EsxB, Ess [ESAT-6 (Early SecretedAntigen Target 6 kDa)] secretion system] extracellular B; Hla,alpha-hemolysin; LukD, Leukocidin D; vWbp, von Willebrand bindingprotein). The values represent the fold increase of samples from mAb 3F6treated animals over the isotype control animal sera samples (n=7 forIgG_(2a), n=8 for 3F6). Data are the means and error bars represent±SEM. Results in A-B are representative of two independent analyses.

FIG. 2: Avidity of protein A specific monoclonal antibodies. Monoclonalantibodies were incubated with increasing concentration (0-4M) ofammonium thiocyanate to perturb the antigen-antibody specificinteraction in (A) IgG₁ isotype monoclonal antibodies, (B) IgG_(2a)isotype monoclonal antibodies and (C) IgG_(2b) isotype monoclonalantibodies. Data are the means and error bars represent ±SEM. Results inA-C are representative of three independent analyses.

FIG. 3: SpA_(KKAA)-specific mAbs bind wild-type protein A. (A) ELISAexamining the binding of immobilized wild-type protein A (SpA) toisotype control antibodies (IgG₁, IgG_(2a) or IgG_(2b)) orSpA_(KKAA)-specific mAbs (5A10, 3F6 and 3D11). (B) Association of horseradish peroxidase (HRP)-conjugated SpA_(KKAA)-specific mAbs (5A10-HRP,3F6-HRP and 3D11-HRP) to immobilized SpA_(KKAA) was examined in a platereader experiment where SpA_(KKAA) was first incubated with isotypecontrol antibodies (IgG₁, IgG_(2a) or IgG_(2b)) or three differentSpA_(KKAA)-specific mAbs (5A10, 3F6 and 3D11) to assess the possibilityof competitive inhibition for antibody that bind the same or closelyrelated sites (n=3). The values at OD_(405nm) were measured andnormalized to the interaction of SpA_(KKAA) and HRP-conjugated SpAspecific mAbs. Data are the means and error bars represent ±SEM. Data inpanels A and B are representative of three independent analyses. Theasterisks denotes statistical significance (P<0.05).

FIG. 4: SpA_(KKAA)-mAbs prevent the association of staphylococcalprotein A with immunoglobulin. (A) Isotype control antibodies orSpA_(KKAA)-mAbs were used to perturb the binding of human IgG towardproteins (wild-type SpA, or variants that lack the ability to bind Fcγ(SpA_(KK)) or Fab (SpA_(AA)) immobilized on ELISA plates. The valueswere normalized to the protein A interaction with human IgG withoutantibodies (n=4). (B) Staphylococci were grown to mid-log phase andincubated with either isotype control antibody or mAb 3F6 and followedby addition of 2 μg wild-type Sbi₁₋₄. Upon incubation, Sbi₁₋₄consumption was measured by immunoblot using affinity purifiedα-SpA_(KKAA) rabbit antibody. The values were normalized to Sbi₁₋₄sedimentation without antibody (No Ab). (C) Affinity purified SpA (200μg) was injected into the peritoneal cavity of mice pre-treated with 85μg (5 mg·kg⁻¹) of either isotype control antibody or mAb 3F6. Animalswere euthanized at indicated time points to measure the amount of SpA incirculating blood by immunoblot with affinity purified α-SpA_(KK)AArabbit antibody (n=3 per time point). The values were normalized to thetotal amount of SpA injected at 0 min. Data are the means and error barsrepresent ±SEM. Results in A-C are representative of two independentanalyses. The asterisks denotes statistical significance (P<0.05).

FIG. 5: SpA_(KKAA)-mAbs promote opsonophagocytic killing of S. aureus inmouse and human blood. (A) Lepirudin anticoagulated mouse blood wasincubated with 5×10⁵ CFU S. aureus Newman in the presence of isotypemouse antibody controls or SpA_(KKAA)-mAbs (2 μg·ml⁻¹) for 30 minutesand survival measured (n=3). (B) Lepirudin anti-coagulated human wholeblood was incubated with 5×10⁶ CFU S. aureus MW2 in the presence ofisotype mouse antibody controls or SpA_(KKAA)-mAbs (10 μg·ml⁻¹) for 120minutes and survival measured (n=3). (C-H) At 60 minutes of incubationof staphylococci in anticoagulated human blood, clusters ofextracellular staphylococci were detected in samples incubated withmouse isotype antibody controls (gray arrowheads), whereas staphylococciwere found within neutrophils (black arrowheads) in samples withSpA_(KKAA)-mAbs. Data are the means and error bars represent ±SEM.Results in A-H are representative of three independent analyses. Theasterisks denotes statistical significance (P<0.05).

FIG. 6: Generation of protein A specific immune response by mAb 3F6.Protein A-specific antibody titers in animals (n=5 per group) that hadreceived a mixture of 20 μg of protein A variants (SpA, SpA_(KK),SpA_(AA), SpA_(KKAA), and PBS) and 85 μg of mAb 3F6 (an IgG2a antibody)or its isotype control were measured by ELISA. Immune titers werenormalized to their isotype control standards. Data are the means anderror bars represent ±SEM. Results are representative of two independentanalyses.

FIG. 7: Interaction of human immunoglobulin fragments with protein Avariants. Association of immobilized protein A variants (wild-type SpA,SpA_(KK), SpA_(AA) or SpA_(KKAA)) with human immunoglobulin (hIgG), aswell as its Fc or F(ab)₂ fragments were analyzed by ELISA and normalizedto the interaction of SpA and human IgG. Statistical significance of SpAvariants were compared against SpA binding to each ligand (human IgG, Fcor F(ab)₂ fragments, n=4). Data are the means and error bars represent±SEM. Results are representative of three independent analyses. Theasterisks denotes statistical significance (P<0.05).

FIG. 8A-B: SpA_(KKAA) mAb CDR alignments. Amino Acid sequences from CDRs(complementarity determining regions) obtained from hybridoma cell lineImmunoglobulin genes were aligned using ClustalW2. An * (asterisk)indicates positions which have a single, fully conserved residue. :(colon) indicates conservation between groups of strongly similarproperties—scoring>0.5 in the Gonnet PAM 250 matrix. . (period)indicates conservation between groups of weakly similarproperties—scoring=<0.5 in the Gonnet PAM 250 matrix. mAb rank based onCFU reduction in the murine renal abscess model appears in superscriptin front of mAb identifier. Mouse IgG isotype is indicated.AVFPMILW—Small (small+hydrophobic (incl.aromatic—Y)), DE—Acidic,RK—Basic-H, STYHCNGQ—Hydroxyl+sulfhydryl+amine+G.

FIG. 9: SpA monoclonal antibody (Spa27) fails to elicit protectiveimmunity in mice. (A) ELISA examining the association SpA-mAb (Spa27)and SpA_(KKAA)-mAb (3F6) with immobilized wild-type protein A (SpA) andvariants lacking immunoglobulin binding via Fcγ (SpA_(KK)), Fab(SpA_(AA)) or Fcγ and Fab (SpA_(KKAA))(n=3). (B) Cohorts of animals(n=9-15) were immunized by intraperitoneal injection with either mock(PBS), Spa27 at 5 mg·kg⁻¹, or 3F6 at 5 or 50 mg·kg⁻¹. Twenty-four hourspost immunization, animals were challenged with 5×10⁶ CFU of S. aureusUSA300. Four days post challenge, animals were euthanized to enumeratethe staphylococcal load in kidneys.

FIG. 10A-E: mAb 358A76.1 specifically recognizes the E domain ofstaphylococcal protein A. ELISA examining the association of (A) mAbs358A76.1 and (B) 3F6 with immobilized non-toxigenic protein A variant(SpA_(KKAA)), each immunoglobulin binding domain (E_(KKAA), D_(KKAA),A_(KKAA), B_(KKAA), and C_(KKAA)), and synthetic linear peptides derivedfrom the three helices (H1, H2, H3, H1+2, H2+3) of the E_(KKAA)immunoglobulin binding domain (IgBD). (C) Alignment of amino acidsequences of the five IgBDs of protein A reveals amino acid residues inE domain that are different from the conserved amino acid residues ofthe remaining four IgBDs (dashed boxes). Amino acid residues substitutedwithin non-toxigenic protein A are identified by gray boxes. (D) Aminoacid sequence homology level was compared using ClustalW and the numbersrepresent the percent of amino acid homology between immunoglobulinbinding domains. (E) The binding of horse radish peroxidase(HRP)-conjugated mAbs (358A76.1-HRP and 3F6-HRP) to SpA_(KKAA)immobilized in an ELISA plate was assessed in a plate reader experimentwhere SpA_(KKAA) was first incubated with isotype control antibody(IgG_(2a)) or mAbs (358A76.1 and 3F6) to identify competitive inhibitionof antibodies that bind the same or closely related sites. Values atOD_(405nm) were recorded and normalized for the interaction ofSpA_(KKAA) and HRP-conjugated SpA specific mAbs.

FIG. 11A-B: SpA monoclonal antibody 358A76.1 fails to elicit protectiveimmunity in mice. (A) Cohorts of animals (n=10) were immunized byintraperitoneal injection with either mock (IgG_(2a) isotype controlmAb), mAb 358A76.1 or mAb 3F6 at 5 mg·kg⁻¹. Twenty-four hours postimmunization, animals were challenged via intravenous inoculation with5×10⁶ CFU of S. aureus USA300. Four days post challenge, animals wereeuthanized to enumerate the staphylococcal load in kidneys. (B)Anti-coagulated mouse blood was incubated with 5×10⁵ CFU S. aureusUSA300 (LAC) in the presence of IgG2a isotype control mAb, mAb 358A76.1or mAb 3F6 (10 μg·ml⁻¹) for 30 minutes; staphylococcal survival wasmeasured. (C) Isotype control antibodies, mAb 358A76.1 or mAb 3F6 wereused to perturb the binding of human IgG to wild-type protein A (SpA)immobilized on ELISA plates. The values were normalized to the protein Ainteraction with human IgG in the absence of antibodies.

DETAILED DESCRIPTION OF THE INVENTION

Staphylococcus aureus is a commensal of the human skin and nares, andthe leading cause of bloodstream, skin and soft tissue infections(Klevens et al., 2007). Recent dramatic increases in the mortality ofstaphylococcal diseases are attributed to the spread ofmethicillin-resistant S. aureus (MRSA) strains often not susceptible toantibiotics (Kennedy et al., 2008). In a large retrospective study, theincidence of MRSA infections was 4.6% of all hospital admissions in theUnited States (Klevens et al., 2007). The annual health care costs for94,300 MRSA infected individuals in the United States exceed $2.4billion (Klevens et al., 2007). The current MRSA epidemic hasprecipitated a public health crisis that needs to be addressed bydevelopment of a preventive vaccine (Boucher and Corey, 2008). To date,an FDA licensed vaccine that prevents S. aureus diseases is notavailable.

The inventors describe here staphylococcal Protein A-binding antibodiesand the antigen binding determinants thereof. In particular, an array ofmonoclonal antibodies have been produced using a SpA mutant protein thatlacks both toxicity (Fcγ interaction) and super antigen activity againstB-cells (Fab interaction). Many of the antibodies were found to interactwith SpA with high affinity and specificity. Importantly, the antibodiesare able to neutralize the molecular mechanisms of staphylococcalprotein A. Further, when administered to animals, the antibodies reducedbacterial load and abscess formation following challenge with virulentS. aureus. Because these molecules are able to block theimmunosuppressive effects of SpA, such antibody may also enhance hostimmune response following staphylococcal infection. Thus, theSpA-binding molecules of the embodiments offer a new and effectiveavenue to treat or prevent staphylococcal disease.

I. SPA POLYPEPTIDES

Certain aspects of the embodiments concern SpA polypeptides, such aswild type SpA provided here as SEQ ID NO: 1. In certain aspect, however,the embodiments concern mutant or variant SpA polypeptides, such aspolypeptides that lacks B-cell super antigen activity and/ornon-specific immunoglobulin binding activity (i.e., binding the Ig thatis not dependent upon the CDR sequence of the Ig). In particular,certain embodiments concern polypeptides (e.g., polypeptides comprisingantibody CDR domains) that specifically bind to a SpA polypeptide thatlacks B-cell super antigen activity and/or non-specific immunoglobulinbinding activity.

The N-terminal part of protein A is comprised of four or five 56-61amino acid residue immunoglobulin binding domains (IgBD A-E); SpAvariants for use according to the embodiments can be, for example, fulllength SpA variant comprising a variant A, B, C, D, and/or E domain. Incertain aspects, the SpA variant comprises or consists of the amino acidsequence that is 80, 90, 95, 98, 99, or 100% identical to the amino acidsequence of SEQ ID NO:7. In other embodiments the SpA variant comprisesa segment of SpA. The SpA segment can comprise at least or at most 1, 2,3, 4, 5 or more IgG binding domains. The IgG domains can be at least orat most 1, 2, 3, 4, 5 or more variant A, B, C, D, or E domains. Incertain aspects the SpA variant comprises at least or at most 1, 2, 3,4, 5, or more variant A domains. In a further aspect the SpA variantcomprises at least or at most 1, 2, 3, 4, 5, or more variant B domains.In still a further aspect the SpA variant comprises at least or at most1, 2, 3, 4, 5, or more variant C domains. In yet a further aspect theSpA variant comprises at least or at most 1, 2, 3, 4, 5, or more variantD domains. In certain aspects the SpA variant comprises at least or atmost 1, 2, 3, 4, 5, or more variant E domains. In a further aspect theSpA variant comprises a combination of A, B, C, D, and E domains invarious combinations and permutations. The combinations can include allor part of a SpA signal peptide segment, a SpA region X segment, and/ora SpA sorting signal segment. In other aspects the SpA variant does notinclude a SpA signal peptide segment, a SpA region X segment, and/or aSpA sorting signal segment. In certain aspects a variant A domaincomprises a substitution at position(s) 7, 8, 34, and/or 35 of SEQ IDNO:4. In another aspect a variant B domain comprises a substitution atposition(s) 7, 8, 34, and/or 35 of SEQ ID NO:6. In still another aspecta variant C domain comprises a substitution at position(s) 7, 8, 34,and/or 35 of SEQ ID NO:5. In certain aspects a variant D domaincomprises a substitution at position(s) 9, 10, 36, and/or 37 of SEQ IDNO:2. In a further aspect a variant E domain comprises a substitution atposition(s) 6, 7, 33, and/or 34 of SEQ ID NO:3.

In certain aspects, an SpA domain D variant or its equivalent cancomprise a mutation at position 9 and 36; 9 and 37; 9 and 10; 36 and 37;10 and 36; 10 and 37; 9, 36, and 37; 10, 36, and 37, 9, 10 and 36; or 9,10 and 37 of SEQ ID NO:2. In a further aspect, analogous mutations canbe included in one or more of domains A, B, C, or E.

In further aspects, the amino acid glutamine (Q) at position 9 of SEQ IDNO:2 (or its analogous amino acid in other SpA domains) can be replacedwith an alanine (A), an asparagine (N), an aspartic acid (D), a cysteine(C), a glutamic acid (E), a phenylalanine (F), a glycine (G), ahistidine (H), an isoleucine (I), a lysine (K), a leucine (L), amethionine (M), a proline (P), a serine (S), a threonine (T), a valine(V), a tryptophane (W), or a tyrosine (Y). In some aspects the glutamineat position 9 can be substituted with an arginine (R). In a furtheraspect, the glutamine at position 9 of SEQ ID NO:2, or its equivalent,can be substituted with a lysine or a glycine. Any 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more of the substitutions can be explicitly excluded.

In another aspect, the amino acid glutamine (Q) at position 10 of SEQ IDNO:2 (or its analogous amino acid in other SpA domains) can be replacedwith an alanine (A), an asparagine (N), an aspartic acid (D), a cysteine(C), a glutamic acid (E), a phenylalanine (F), a glycine (G), ahistidine (H), an isoleucine (I), a lysine (K), a leucine (L), amethionine (M), a proline (P), a serine (S), a threonine (T), a valine(V), a tryptophane (W), or a tyrosine (Y). In some aspects the glutamineat position 10 can be substituted with an arginine (R). In a furtheraspect, the glutamine at position 10 of SEQ ID NO:2, or its equivalent,can be substituted with a lysine or a glycine. Any 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more of the substitutions can be explicitly excluded.

In certain aspects, the aspartic acid (D) at position 36 of SEQ ID NO:2(or its analogous amino acid in other SpA domains) can be replaced withan alanine (A), an asparagine (N), an arginine (R), a cysteine (C), aphenylalanine (F), a glycine (G), a histidine (H), an isoleucine (I), alysine (K), a leucine (L), a methionine (M), a proline (P), a glutamine(Q), a serine (S), a threonine (T), a valine (V), a tryptophane (W), ora tyrosine (Y). In some aspects the aspartic acid at position 36 can besubstituted with a glutamic acid (E). In certain aspects, an asparticacid at position 36 of SEQ ID NO:2, or its equivalent, can besubstituted with an alanine or a serine. Any 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more of the substitutions can be explicitly excluded.

In another aspect, the aspartic acid (D) at position 37 of SEQ ID NO:2(or its analogous amino acid in other SpA domains) can be replaced withan alanine (A), a an asparagine (N), an arginine (R), a cysteine (C), aphenylalanine (F), a glycine (G), a histidine (H), an isoleucine (I), alysine (K), a leucine (L), a methionine (M), a proline (P), a glutamine(Q), a serine (S), a threonine (T), a valine (V), a tryptophane (W), ora tyrosine (Y). In some aspects the aspartic acid at position 37 can besubstituted with a glutamic acid (E). In certain aspects, an asparticacid at position 37 of SEQ ID NO:2, or its equivalent, can besubstituted with an alanine or a serine. Any 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more of the substitutions can be explicitly excluded.

In a particular embodiment the amino at position 9 of SEQ ID NO:2 (or ananalogous amino acid in another SpA domain) is replaced by an alanine(A), a glycine (G), an isoleucine (I), a leucine (L), a proline (P), aserine (S), or a valine (V), In certain aspects the amino acid atposition 9 of SEQ ID NO:2 is replaced by a glycine. In a further aspectthe amino acid at position 9 of SEQ ID NO:2 is replaced by a lysine.

In a particular embodiment the amino at position 10 of SEQ ID NO:2 (oran analogous amino acid in another SpA domain) is replaced by an alanine(A), a glycine (G), an isoleucine (I), a leucine (L), a proline (P), aserine (S), or a valine (V), In certain aspects the amino acid atposition 10 of SEQ ID NO:2 is replaced by a glycine. In a further aspectthe amino acid at position 10 of SEQ ID NO:2 is replaced by a lysine.

In a particular embodiment the amino at position 36 of SEQ ID NO:2 (oran analogous amino acid in another SpA domain) is replaced by an alanine(A), a glycine (G), an isoleucine (I), a leucine (L), a proline (P), aserine (S), or a valine (V), In certain aspects the amino acid atposition 36 of SEQ ID NO:2 is replaced by a serine. In a further aspectthe amino acid at position 36 of SEQ ID NO:2 is replaced by an alanine.

In a particular embodiment the amino at position 37 of SEQ ID NO:2 (oran analogous amino acid in another SpA domain) is replaced by an alanine(A), a glycine (G), an isoleucine (I), a leucine (L), a proline (P), aserine (S), or a valine (V), In certain aspects the amino acid atposition 37 of SEQ ID NO:2 is replaced by a serine. In a further aspectthe amino acid at position 37 of SEQ ID NO:2 is replaced by an alanine.

In certain aspects the SpA variant includes a substitution of (a) one ormore amino acid substitution in an IgG Fc binding sub-domain of SpAdomain A, B, C, D, and/or E that disrupts or decreases binding to IgGFc, and (b) one or more amino acid substitution in a V_(H)3 bindingsub-domain of SpA domain A, B, C, D, and/or E that disrupts or decreasesbinding to V_(H)3. In still further aspects the amino acid sequence of aSpA variant comprises an amino acid sequence that is at least 50%, 60%,70%, 80%, 90%, 95%, or 100% identical, including all values and rangesthere between, to the amino acid sequence of SEQ ID NOs:2-6.

In a further aspect the SpA variant includes (a) one or more amino acidsubstitution in an IgG Fc binding sub-domain of SpA domain D, or at acorresponding amino acid position in other IgG domains, that disrupts ordecreases binding to IgG Fc, and (b) one or more amino acid substitutionin a V_(H)3 binding sub-domain of SpA domain D, or at a correspondingamino acid position in other IgG domains, that disrupts or decreasesbinding to V_(H)3. In certain aspects amino acid residue F5, Q9, Q10,S11, F13, Y14, L17, N28, I31, and/or K35 (SEQ ID NO:2,QQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNES) of the IgG Fcbinding sub-domain of domain D are modified or substituted. In certainaspects amino acid residue Q26, G29, F30, S33, D36, D37, Q40, N43,and/or E47 (SEQ ID NO:2) of the V_(H)3 binding sub-domain of domain Dare modified or substituted such that binding to Fc or V_(H)3 isattenuated. In further aspects corresponding modifications orsubstitutions can be engineered in corresponding positions of the domainA, B, C, and/or E. Corresponding positions are defined by alignment ofthe domain D amino acid sequence with one or more of the amino acidsequences from other IgG binding domains of SpA. In certain aspects theamino acid substitution can be any of the other 20 amino acids. In afurther aspect conservative amino acid substitutions can be specificallyexcluded from possible amino acid substitutions. In other aspects onlynon-conservative substitutions are included. In any event, anysubstitution or combination of substitutions that reduces the binding ofthe domain such that SpA toxicity is significantly reduced iscontemplated. The significance of the reduction in binding refers to avariant that produces minimal to no toxicity when introduced into asubject and can be assessed using in vitro methods described herein.

In certain embodiments, a variant SpA comprises at least or at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more variant SpA domain D peptides. Incertain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, or 19 or more amino acid residues of the variant SpA aresubstituted or modified—including but not limited to amino acids F5, Q9,Q10, S11, F13, Y14, L17, N28, I31, and/or K35 (SEQ ID NO:2) of the IgGFc binding sub-domain of domain D and amino acid residue Q26, G29, F30,S33, D36, D37, Q40, N43, and/or E47 (SEQ ID NO:2) of the V_(H)3 bindingsub-domain of domain D. In one aspect glutamine residues at position 9and/or 10 of SEQ ID NO:2 (or corresponding positions in other domains)are mutated. In another aspect, aspartic acid residues 36 and/or 37 ofSEQ ID NO:2 (or corresponding positions in other domains) are mutated.In a further aspect, glutamine 9 and 10, and aspartic acid residues 36and 37 are mutated. Purified non-toxigenic SpA or SpA-D mutants/variantsdescribed herein are no longer able to significantly bind (i.e.,demonstrate attenuated or disrupted binding affinity) Fcγ or F(ab)₂V_(H)3 and also do not stimulate B cell apoptosis.

It is contemplated that cariants of SpA may also include the samevariations in domains A, B, C, and/or E as in domain D described above.In some embodiments, a SpA binding polypeptide or antibody may bind to aSpA variant that has a KKAA variation described herein in each ofdomains A, B, C, D, and E. In further embodiments, that same SpA bindingpolypeptide or antibody may also bind to a variant that has a GGSSvariation instead of the KKAA in every domain. Additionally, in certainembodiments, a SpA binding polypeptide or antibody may bind to a variantSbi antigen that is altered with respect to one or more of its domainslike in SpA. An example of this is shown in FIG. 4.

Moreover, it is contemplated that SpA binding polypeptides or antibodiesdescribed herein may be capable of competing with SpA binding forimmunoglobulin Fc or Fab region or hinder SpA disruption ofimmunoglobulin function. Also it is contemplated that SpA bindingpolypeptides or antibodies described herein may or be capable ofperturbing SpA disruption of immunoglobulin function or SpA binding toimmunoglobulin Fc or Fab region. In certain embodiments, thisproperty(ies) allows the therapeutic compound to be used to treatinfection. Furthermore, methods involve a SpA binding polypeptide orantibody that is capable of neutralizing SpA disruption ofimmunoglobulin function or SpA binding to immunoglobulin Fc or Fabregion.

Non-toxigenic Protein A variants can be used as subunit vaccines andraise humoral immune responses and confer protective immunity against S.aureus challenge. Compared to wild-type full-length Protein A or thewild-type SpA-domain D, immunization with SpA-D variants resulted in anincrease in Protein A specific antibody. Further SpA variants andmethods for using the same are provided in PCT Publication No. WO2011/005341 and PCT Appln. No. PCT/US 11/42845, both incorporated hereinby reference.

II. PROTEINACEOUS COMPOSITIONS

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, with or withoutthe loss of other functions or properties. Substitutions may beconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine. Alternatively, substitutions may benon-conservative such that a function or activity of the polypeptide isaffected. Non-conservative changes typically involve substituting aresidue with one that is chemically dissimilar, such as a polar orcharged amino acid for a nonpolar or uncharged amino acid, and viceversa.

Proteins may be recombinant, or synthesized in vitro. Alternatively, anon-recombinant or recombinant protein may be isolated from bacteria. Itis also contemplated that a bacteria containing such a variant may beimplemented in compositions and methods. Consequently, a protein neednot be isolated.

The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, and also refers to codons that encode biologicallyequivalent amino acids (see Table, below).

Codon Table Amino Acids Codons Alanine Ala A GCA GCC GCG GCU CysteineCys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU ThreonineThr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids, or 5′ or 3′ sequences, respectively, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression isconcerned. The addition of terminal sequences particularly applies tonucleic acid sequences that may, for example, include various non-codingsequences flanking either of the 5′ or 3′ portions of the coding region.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andin its underlying DNA coding sequence, and nevertheless produce aprotein with like properties. It is thus contemplated by the inventorsthat various changes may be made in the DNA sequences of genes withoutappreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. It is understood that an amino acid can besubstituted for another having a similar hydrophilicity value and stillproduce a biologically equivalent and immunologically equivalentprotein.

As outlined above, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known and include: arginine andlysine; glutamate and aspartate; serine and threonine; glutamine andasparagine; and valine, leucine and isoleucine.

It is contemplated that in compositions there is between about 0.001 mgand about 10 mg of total polypeptide, peptide, and/or protein per ml.Thus, the concentration of protein in a composition can be about, atleast about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or anyrange derivable therein). Of this, about, at least about, or at mostabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100% may be an antibody that binds SpA,and may be used in combination with other staphylococcal proteins orprotein-binding antibodies described herein.

A. Polypeptides and Polypeptide Production

Embodiments involve polypeptides, peptides, and proteins and immunogenicfragments thereof for use in various aspects described herein. Forexample, specific antibodies are assayed for or used in neutralizing orinhibiting Staphylococcal infection. In specific embodiments, all orpart of proteins described herein can also be synthesized in solution oron a solid support in accordance with conventional techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols. See, for example, Stewart and Young,(1984); Tam et al., (1983); Merrifield, (1986); and Barany andMerrifield (1979), each incorporated herein by reference. Alternatively,recombinant DNA technology may be employed wherein a nucleotide sequencethat encodes a peptide or polypeptide is inserted into an expressionvector, transformed or transfected into an appropriate host cell andcultivated under conditions suitable for expression.

One embodiment includes the use of gene transfer to cells, includingmicroorganisms, for the production and/or presentation of proteins. Thegene for the protein of interest may be transferred into appropriatehost cells followed by culture of cells under the appropriateconditions. A nucleic acid encoding virtually any polypeptide may beemployed. The generation of recombinant expression vectors, and theelements included therein, are discussed herein. Alternatively, theprotein to be produced may be an endogenous protein normally synthesizedby the cell used for protein production.

In a certain aspects an immunogenic SpA fragment comprises substantiallyall of the extracellular domain of a protein which has at least 85%identity, at least 90% identity, at least 95% identity, or at least97-99% identity, including all values and ranges there between, to asequence selected over the length of the fragment sequence.

Also included in immunogenic compositions are fusion proteins composedof Staphylococcal proteins, or immunogenic fragments of staphylococcalproteins (e.g., SpA). Alternatively, embodiments also include individualfusion proteins of Staphylococcal proteins or immunogenic fragmentsthereof, as a fusion protein with heterologous sequences such as aprovider of T-cell epitopes or purification tags, for example:β-galactosidase, glutathione-S-transferase, green fluorescent proteins(GFP), epitope tags such as FLAG, myc tag, poly histidine, or viralsurface proteins such as influenza virus haemagglutinin, or bacterialproteins such as tetanus toxoid, diphtheria toxoid, CRMI97.

B. Antibodies and Antibody-Like Molecules

In certain aspects, one or more antibodies or antibody-like molecules(e.g., polypeptides comprising antibody CDR domains) may be obtained orproduced which have a specificity for an SpA. These antibodies may beused in various diagnostic or therapeutic applications described herein.

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE as well aspolypeptides comprising antibody CDR domains that retain antigen bindingactivity. Thus, the term “antibody” is used to refer to anyantibody-like molecule that has an antigen binding region, and includesantibody fragments such as Fab′, Fab, F(ab′)₂, single domain antibodies(DABs), Fv, scFv (single chain Fv), and polypeptides with antibody CDRs,scaffolding domains that display the CDRs (e.g., anticalins) or ananobody. For example, the nanobody can be antigen-specific VHH (e.g., arecombinant VHH) from a camelid IgG2 or IgG3, or a CDR-displaying framefrom such camelid Ig. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

“Mini-antibodies” or “minibodies” are also contemplated for use withembodiments. Minibodies are sFv polypeptide chains which includeoligomerization domains at their C-termini, separated from the sFv by ahinge region. Pack et al. (1992). The oligomerization domain comprisesself-associating α-helices, e.g., leucine zippers, that can be furtherstabilized by additional disulfide bonds. The oligomerization domain isdesigned to be compatible with vectorial folding across a membrane, aprocess thought to facilitate in vivo folding of the polypeptide into afunctional binding protein. Generally, minibodies are produced usingrecombinant methods well known in the art. See, e.g., Pack et al.(1992); Cumber et al. (1992).

Antibody-like binding peptidomimetics are also contemplated inembodiments. Liu et al. (2003) describe “antibody like bindingpeptidomimetics” (ABiPs), which are peptides that act as pared-downantibodies and have certain advantages of longer serum half-life as wellas less cumbersome synthesis methods.

Alternative scaffolds for antigen binding peptides, such as CDRs arealso available and can be used to generate SpA-binding molecules inaccordance with the embodiments. Generally, a person skilled in the artknows how to determine the type of protein scaffold on which to graft atleast one of the CDRs arising from the original antibody. Moreparticularly, it is known that to be selected such scaffolds must meetthe greatest number of criteria as follows (Skerra, 2000): goodphylogenetic conservation; known three-dimensional structure (as, forexample, by crystallography, NMR spectroscopy or any other techniqueknown to a person skilled in the art); small size; few or nopost-transcriptional modifications; and/or easy to produce, express andpurify.

The origin of such protein scaffolds can be, but is not limited to, thestructures selected among: fibronectin and preferentially fibronectintype III domain 10, lipocalin, anticalin (Skerra, 2001), protein Zarising from domain B of protein A of Staphylococcus aureus, thioredoxinA or proteins with a repeated motif such as the “ankyrin repeat” (Kohlet al., 2003), the “armadillo repeat”, the “leucine-rich repeat” and the“tetratricopeptide repeat”. For example, anticalins or lipocalinderivatives are a type of binding proteins that have affinities andspecificities for various target molecules and can be used as SpAbinding molecules. Such proteins are described in US Patent PublicationNos. 20100285564, 20060058510, 20060088908, 20050106660, and PCTPublication No. WO2006/056464, incorporated herein by reference.

Scaffolds derived from toxins such as, for example, toxins fromscorpions, insects, plants, mollusks, etc., and the protein inhibitersof neuronal NO synthase (PIN) may also be used in certain aspects.

Monoclonal antibodies (MAbs) are recognized to have certain advantages,e.g., reproducibility and large-scale production. Embodiments includemonoclonal antibodies of the human, murine, monkey, rat, hamster, rabbitand chicken origin.

“Humanized” antibodies are also contemplated, as are chimeric antibodiesfrom mouse, rat, or other species, bearing human constant and/orvariable region domains, bispecific antibodies, recombinant andengineered antibodies and fragments thereof. As used herein, the term“humanized” immunoglobulin refers to an immunoglobulin comprising ahuman framework region and one or more CDR's from a non-human (usually amouse or rat) immunoglobulin. The non-human immunoglobulin providing theCDR's is called the “donor” and the human immunoglobulin providing theframework is called the “acceptor”. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin. In order to describe antibodies of some embodiments, thestrength with which an antibody molecule binds an epitope, known asaffinity, can be measured. The affinity of an antibody may be determinedby measuring an association constant (K_(a)) or dissociation constant(K_(d)). Antibodies deemed useful in certain embodiments may have anassociation constant of about, at least about, or at most about 10⁶,10⁷, 10⁸, 10⁹ or 10¹⁰ M or any range derivable therein. Similarly, insome embodiments antibodies may have a dissociation constant of about,at least about or at most about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or 10⁻¹⁰. M orany range derivable therein. These values are reported for antibodiesdiscussed herein and the same assay may be used to evaluate the bindingproperties of such antibodies

In certain embodiments, a polypeptide that specifically binds to SpA isable to neutralize protein A and/or promote opsonophagocytic killing ofstaphylococci. Moreover, in some embodiments, the polypeptide that isused can provided protective immunity against S. aureus disease. It iscontemplated that mAb 358A76.1 is excluded from these embodiments.

1. Methods for Generating Antibodies

Methods for generating antibodies (e.g., monoclonal antibodies and/ormonoclonal antibodies) are known in the art. Briefly, a polyclonalantibody is prepared by immunizing an animal with a SpA polypeptide(e.g., a non-toxogenic SpA) or a portion thereof in accordance withembodiments and collecting antisera from that immunized animal.

A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. The choice ofanimal may be decided upon the ease of manipulation, costs or thedesired amount of sera, as would be known to one of skill in the art. Itwill be appreciated that antibodies can also be produced transgenicallythrough the generation of a mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies can beproduced in, and recovered from, the milk of goats, cows, or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and5,741,957.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitableadjuvants include any acceptable immunostimulatory compound, such ascytokines, chemokines, cofactors, toxins, plasmodia, syntheticcompositions or vectors encoding such adjuvants.

Adjuvants that may be used in accordance with embodiments include, butare not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, -interferon, GMCSP,BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP,CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, whichcontains three components extracted from bacteria, MPL, trehalosedimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80emulsion is also contemplated. MHC antigens may even be used. Exemplaryadjuvants may include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and/or aluminum hydroxideadjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or downregulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA);low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, NJ), cytokinessuch as -interferon, IL-2, or IL-12 or genes encoding proteins involvedin immune helper functions, such as B-7.

The amount of immunogen composition used in the production of antibodiesvaries upon the nature of the immunogen as well as the animal used forimmunization. A variety of routes can be used to administer theimmunogen including but not limited to subcutaneous, intramuscular,intradermal, intraepidermal, intravenous and intraperitoneal. Theproduction of antibodies may be monitored by sampling blood of theimmunized animal at various points following immunization.

A second, booster dose (e.g., provided in an injection), may also begiven. The process of boosting and titering is repeated until a suitabletiter is achieved. When a desired level of immunogenicity is obtained,the immunized animal can be bled and the serum isolated and stored,and/or the animal can be used to generate MAbs.

For production of rabbit polyclonal antibodies, the animal can be bledthrough an ear vein or alternatively by cardiac puncture. The removedblood is allowed to coagulate and then centrifuged to separate serumcomponents from whole cells and blood clots. The serum may be used as isfor various applications or else the desired antibody fraction may bepurified by well-known methods, such as affinity chromatography usinganother antibody, a peptide bound to a solid matrix, or by using, e.g.,protein A or protein G chromatography, among others.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified protein, polypeptide, peptide or domain, be it awild-type or mutant composition. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells.

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies. Insome embodiments, Rodents such as mice and rats are used in generatingmonoclonal antibodies. In some embodiments, rabbit, sheep or frog cellsare used in generating monoclonal antibodies. The use of rats is wellknown and may provide certain advantages (Goding, 1986, pp. 60 61). Mice(e.g., BALB/c mice) are routinely used and generally give a highpercentage of stable fusions.

The animals are injected with antigen, generally as described above. Theantigen may be mixed with adjuvant, such as Freund's complete orincomplete adjuvant. Booster administrations with the same antigen orDNA encoding the antigen may occur at approximately two-week intervals.As discussed in the Examples, the antigen may be altered compared to anantigen sequence found in nature. In some embodiments, a variant oraltered Protein A peptide or polypeptide is employed to generateantibodies. In certain embodiments, the SpA variant has 1, 2, 3, 4, 5,6, 7, or 8 changes in 1, 2, 3, 4, or all 5 of the A, B, C, D, or Edomains of SpA.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Generally, spleen cells are a rich source of antibody-producingcells that are in the dividing plasmablast stage. Typically, peripheralblood cells may be readily obtained, as peripheral blood is easilyaccessible.

In some embodiments, a panel of animals will have been immunized and thespleen of an animal with the highest antibody titer will be removed andthe spleen lymphocytes obtained by homogenizing the spleen with asyringe. Typically, a spleen from an immunized mouse containsapproximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma producing fusion procedures preferably are non antibodyproducing, have high fusion efficiency, and enzyme deficiencies thatrender then incapable of growing in certain selective media whichsupport the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65 66, 1986; Campbell, pp. 75 83,1984). cites). For example, where the immunized animal is a mouse, onemay use P3 X63/Ag8, X63 Ag8.653, NS1/1.Ag 41, Sp210 Ag14, FO, NSO/U, MPC11, MPC11 X45 GTG 1.7 and S194/5XX0 Bul; for rats, one may useR210.RCY3, Y3 Ag 1.2.3, IR983F and 4B210; and U 266, GM1500 GRG2, LICRLON HMy2 and UC729 6 are all useful in connection with human cellfusions. See Yoo et al. (2002), for a discussion of myeloma expressionsystems.

One murine myeloma cell is the NS-1 myeloma cell line (also termedP3-NS-1-Ag4-1), which is readily available from the NIGMS Human GeneticMutant Cell Repository by requesting cell line repository number GM3573.Another mouse myeloma cell line that may be used is the 8 azaguanineresistant mouse murine myeloma SP2/0 non producer cell line.

Methods for generating hybrids of antibody producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al., (1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp.7174, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

A selection medium is HAT. Only cells capable of operating nucleotidesalvage pathways are able to survive in HAT medium. The myeloma cellsare defective in key enzymes of the salvage pathway, e.g., hypoxanthinephosphoribosyl transferase (HPRT), and they cannot survive. The B cellscan operate this pathway, but they have a limited life span in cultureand generally die within about two weeks. Therefore, the only cells thatcan survive in the selective media are those hybrids formed from myelomaand B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. First, a sample of the hybridomacan be injected (often into the peritoneal cavity) into ahistocompatible animal of the type that was used to provide the somaticand myeloma cells for the original fusion (e.g., a syngeneic mouse).Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can then be tapped to provideMAbs in high concentration. Second, the individual cell lines could becultured in vitro, where the MAbs are naturally secreted into theculture medium from which they can be readily obtained in highconcentrations.

Further, expression of antibodies (or other moieties therefrom) fromproduction cell lines can be enhanced using a number of knowntechniques. For example, the glutamine synthetase and DHFR geneexpression systems are common approaches for enhancing expression undercertain conditions. High expressing cell clones can be identified usingconventional techniques, such as limited dilution cloning and Microdroptechnology. The GS system is discussed in whole or part in connectionwith European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 andEuropean Patent Application No. 89303964.4.

MAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Fragments of the monoclonal antibodiescan be obtained from the monoclonal antibodies so produced by methodswhich include digestion with enzymes, such as pepsin or papain, and/orby cleavage of disulfide bonds by chemical reduction. Alternatively,monoclonal antibody fragments can be synthesized using an automatedpeptide synthesizer.

It is also contemplated that a molecular cloning approach may be used togenerate monoclonal antibodies. In one embodiment, combinatorialimmunoglobulin phagemid libraries are prepared from RNA isolated fromthe spleen of the immunized animal, and phagemids expressing appropriateantibodies are selected by panning using cells expressing the antigenand control cells. The advantages of this approach over conventionalhybridoma techniques are that approximately 104 times as many antibodiescan be produced and screened in a single round, and that newspecificities are generated by H and L chain combination which furtherincreases the chance of finding appropriate antibodies.

Another embodiment concerns producing antibodies, for example, as isfound in U.S. Pat. No. 6,091,001, which describes methods to produce acell expressing an antibody from a genomic sequence of the cellcomprising a modified immunoglobulin locus using Cre-mediatedsite-specific recombination is disclosed. The method involves firsttransfecting an antibody-producing cell with a homology-targeting vectorcomprising a lox site and a targeting sequence homologous to a first DNAsequence adjacent to the region of the immunoglobulin loci of thegenomic sequence which is to be converted to a modified region, so thefirst lox site is inserted into the genomic sequence via site-specifichomologous recombination. Then the cell is transfected with alox-targeting vector comprising a second lox site suitable forCre-mediated recombination with the integrated lox site and a modifyingsequence to convert the region of the immunoglobulin loci to themodified region. This conversion is performed by interacting the loxsites with Cre in vivo, so that the modifying sequence inserts into thegenomic sequence via Cre-mediated site-specific recombination of the loxsites.

Alternatively, monoclonal antibody fragments can be synthesized using anautomated peptide synthesizer, or by expression of full-length gene orof gene fragments in E. coli.

It is further contemplated that monoclonal antibodies may be furtherscreened or optimized for properties relating to specificity, avidity,half-life, immunogenicity, binding association, binding disassociation,or overall functional properties relative to being a treatment forinfection. Thus, it is contemplated that monoclonal antibodies may have1, 2, 3, 4, 5, 6, or more alterations in the amino acid sequence of 1,2, 3, 4, 5, or 6 CDRs of monoclonal antibodies 5A10, 8E2, 3A6, 3F6,1F10, 6D11, 3D11, 5A11, 1B10 or 4C1. It is contemplated that the aminoacid in position 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of CDR1, CDR2, CDR3,CDR4, CDR5, or CDR6 of the VJ or VDJ region of the light or heavyvariable region of monoclonal antibodies 5A10, 8E2, 3A6, 3F6, 1F10,6D11, 3D11, 5A11, 1B10 or 4C1 may have an insertion, deletion, orsubstitution with a conserved or non-conserved amino acid. Such aminoacids that can either be substituted or constitute the substitution aredisclosed above.

In some embodiments, fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment constituted with the VL, VH, CL and CH1 domains; (ii) theFd fragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconstituted with the VL and VH domains of a single antibody; (iv) thedAb fragment (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003),which is constituted with a VH or a VL domain; (v) isolated CDR regions;(vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fabfragments (vii) single chain Fv molecules (scFv), wherein a VH domainand a VL domain are linked by a peptide linker which allows the twodomains to associate to form an antigen binding site (Bird et al., 1988;Huston et al., 1988); (viii) bispecific single chain Fv dimers(PCT/US92/09965) and (ix) “diabodies”, multivalent or multispecificfragments constructed by gene fusion (WO94/13804; Holliger et al.,1993). Fv, scFv or diabody molecules may be stabilized by theincorporation of disulphide bridges linking the VH and VL domains(Reiter et al., 1996). Minibodies comprising a scFv joined to a CH3domain may also be made (Hu et al. 1996). The citations in thisparagraph are all incorporated by reference.

Antibodies also include bispecific antibodies. Bispecific orbifunctional antibodies form a second generation of monoclonalantibodies in which two different variable regions are combined in thesame molecule (Holliger, P. & Winter, G. 1999 Cancer and metastasis rev.18:411-419, 1999). Their use has been demonstrated both in thediagnostic field and in the therapy field from their capacity to recruitnew effector functions or to target several molecules on the surface oftumor cells. Where bispecific antibodies are to be used, these may beconventional bispecific antibodies, which can be manufactured in avariety of ways (Holliger et al, PNAS USA 90:6444-6448, 1993), e.g.prepared chemically or from hybrid hybridomas, or may be any of thebispecific antibody fragments mentioned above. These antibodies can beobtained by chemical methods (Glennie et al., 1987 J. Immunol. 139,2367-2375; Repp et al., J. Hemat. 377-382, 1995) or somatic methods(Staerz U. D. and Bevan M. J. PNAS 83, 1986; et al., Method Enzymol.121:210-228, 1986) but likewise by genetic engineering techniques whichallow the heterodimerization to be forced and thus facilitate theprocess of purification of the antibody sought (Merchand et al. NatureBiotech, 16:677-681, 1998). Examples of bispecific antibodies includethose of the BiTE™ technology in which the binding domains of twoantibodies with different specificity can be used and directly linkedvia short flexible peptides. This combines two antibodies on a shortsingle polypeptide chain. Diabodies and scFv can be constructed withoutan Fc region, using only variable domains, potentially reducing theeffects of anti-idiotypic reaction. The citations in this paragraph areall incorporated by reference.

Bispecific antibodies can be constructed as entire IgG, as bispecificFab′2, as Fab′PEG, as diabodies or else as bispecific scFv. Further, twobispecific antibodies can be linked using routine methods known in theart to form tetravalent antibodies.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against SpA, then a library can be made where the other arm isvaried and an antibody of appropriate specificity selected. Bispecificwhole antibodies may be made by alternative engineering methods asdescribed in Ridgeway et al, (Protein Eng., 9:616-621, 1996), which ishereby incorporated by reference.

C. Antibody and Polypeptide Conjugates

Embodiments provide antibodies and antibody-like molecules against SpAproteins, polypeptides and peptides that are linked to at least oneagent to form an antibody conjugate or payload. In order to increase theefficacy of antibody molecules as diagnostic or therapeutic agents, itis conventional to link or covalently bind or complex at least onedesired molecule or moiety. Such a molecule or moiety may be, but is notlimited to, at least one effector or reporter molecule. Effectormolecules comprise molecules having a desired activity, e.g., cytotoxicactivity. Non-limiting examples of effector molecules which have beenattached to antibodies include toxins, therapeutic enzymes, antibiotics,radio-labeled nucleotides and the like. By contrast, a reporter moleculeis defined as any moiety which may be detected using an assay.Non-limiting examples of reporter molecules which have been conjugatedto antibodies include enzymes, radiolabels, haptens, fluorescent labels,phosphorescent molecules, chemiluminescent molecules, chromophores,luminescent molecules, photoaffinity molecules, colored particles orligands, such as biotin.

Certain examples of antibody conjugates are those conjugates in whichthe antibody is linked to a detectable label. “Detectable labels” arecompounds and/or elements that can be detected due to their specificfunctional properties, and/or chemical characteristics, the use of whichallows the antibody to which they are attached to be detected, and/orfurther quantified if desired. A

Antibody conjugates are generally preferred for use as diagnosticagents. Antibody diagnostics generally fall within two classes, thosefor use in in vitro diagnostics, such as in a variety of immunoassays,and/or those for use in vivo diagnostic protocols, generally known as“antibody directed imaging”. Many appropriate imaging agents are knownin the art, as are methods for their attachment to antibodies (see, fore.g., U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, eachincorporated herein by reference). The imaging moieties used can beparamagnetic ions; radioactive isotopes; fluorochromes; NMR-detectablesubstances; X-ray imaging.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might use astatine²¹¹, carbon¹⁴, chromium⁵¹,chlorine³⁶, cobalt⁵⁷, cobalt⁵⁸, copper⁶⁷, Eu¹⁵², gallium⁶⁷, hydroge³n,iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, iron⁵⁹, phosphorus³²,rhenium¹⁸⁶, rhenium¹⁸⁸, selenium⁷⁵, sulphu³⁵r, technicium^(99m) and/oryttrium⁹⁰. ¹²⁵I is often used in certain embodiments, andtechnicium^(99m) and/or indium¹¹¹ are also often used due to their lowenergy and suitability for long range detection. Radioactively labeledmonoclonal antibodies may be produced according to well-known methods inthe art. For instance, monoclonal antibodies can be iodinated by contactwith sodium and/or potassium iodide and a chemical oxidizing agent suchas sodium hypochlorite, or an enzymatic oxidizing agent, such aslactoperoxidase. Monoclonal antibodies may be labeled with technetium99mby ligand exchange process, for example, by reducing pertechnate withstannous solution, chelating the reduced technetium onto a Sephadexcolumn and applying the antibody to this column. Alternatively, directlabeling techniques may be used, e.g., by incubating pertechnate, areducing agent such as SNCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the antibody. Intermediary functional groupswhich are often used to bind radioisotopes which exist as metallic ionsto antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetracetic acid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed, among others.

Antibody conjugates include those intended primarily for use in vitro,where the antibody is linked to a secondary binding ligand and/or to anenzyme (an enzyme tag) that will generate a colored product upon contactwith a chromogenic substrate. Examples of suitable enzymes include, butare not limited to, urease, alkaline phosphatase, (horseradish) hydrogenperoxidase or glucose oxidase. Preferred secondary binding ligands arebiotin and/or avidin and streptavidin compounds. The use of such labelsis well known to those of skill in the art and are described, forexample, in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241; each incorporated herein byreference.

Yet another known method of site-specific attachment of molecules toantibodies comprises the reaction of antibodies with hapten-basedaffinity labels. Essentially, hapten-based affinity labels react withamino acids in the antigen binding site, thereby destroying this siteand blocking specific antigen reaction. However, this may not beadvantageous since it results in loss of antigen binding by the antibodyconjugate.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3-6-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein byreference). Monoclonal antibodies may also be reacted with an enzyme inthe presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate. In U.S.Pat. No. 4,938,948, imaging of breast tumors is achieved usingmonoclonal antibodies and the detectable imaging moieties are bound tothe antibody using linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In some embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

In some embodiments, anti-SpA antibodies are linked to semiconductornanocrystals such as those described in U.S. Pat. Nos. 6,048,616;5,990,479; 5,690,807; 5,505,928; 5,262,357 (all of which areincorporated herein in their entireties); as well as PCT Publication No.99/26299 (published May 27, 1999). In particular, exemplary materialsfor use as semiconductor nanocrystals in the biological and chemicalassays include, but are not limited to, those described above, includinggroup II-VI, III-V and group IV semiconductors such as ZnS, ZnSe, ZnTe,CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS,BaSe, BaTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlSb, PbS,PbSe, Ge and Si and ternary and quaternary mixtures thereof. Methods forlinking semiconductor nanocrystals to antibodies are described in U.S.Pat. Nos. 6,630,307 and 6,274,323.

III. NUCLEIC ACIDS

In certain embodiments, there are recombinant polynucleotides encodingthe proteins, polypeptides, or peptides described herein. Polynucleotidesequences contemplated include those encoding antibodies to SpA or SpAbinding portions thereof.

As used in this application, the term “polynucleotide” refers to anucleic acid molecule that either is recombinant or has been isolatedfree of total genomic nucleic acid. Included within the term“polynucleotide” are oligonucleotides (nucleic acids 100 residues orless in length), recombinant vectors, including, for example, plasmids,cosmids, phage, viruses, and the like. Polynucleotides include, incertain aspects, regulatory sequences, isolated substantially away fromtheir naturally occurring genes or protein encoding sequences.Polynucleotides may be single-stranded (coding or antisense) ordouble-stranded, and may be RNA, DNA (genomic, cDNA or synthetic),analogs thereof, or a combination thereof. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide.

In this respect, the term “gene,” “polynucleotide,” or “nucleic acid” isused to refer to a nucleic acid that encodes a protein, polypeptide, orpeptide (including any sequences required for proper transcription,post-translational modification, or localization). As will be understoodby those in the art, this term encompasses genomic sequences, expressioncassettes, cDNA sequences, and smaller engineered nucleic acid segmentsthat express, or may be adapted to express, proteins, polypeptides,domains, peptides, fusion proteins, and mutants. A nucleic acid encodingall or part of a polypeptide may contain a contiguous nucleic acidsequence encoding all or a portion of such a polypeptide. It also iscontemplated that a particular polypeptide may be encoded by nucleicacids containing variations having slightly different nucleic acidsequences but, nonetheless, encode the same or substantially similarprotein (see above).

In particular embodiments, there are isolated nucleic acid segments andrecombinant vectors incorporating nucleic acid sequences that encode apolypeptide (e.g., an antibody or fragment thereof) that binds to SpA.The term “recombinant” may be used in conjunction with a polypeptide orthe name of a specific polypeptide, and this generally refers to apolypeptide produced from a nucleic acid molecule that has beenmanipulated in vitro or that is a replication product of such amolecule.

The nucleic acid segments, regardless of the length of the codingsequence itself, may be combined with other nucleic acid sequences, suchas promoters, polyadenylation signals, additional restriction enzymesites, multiple cloning sites, other coding segments, and the like, suchthat their overall length may vary considerably. It is thereforecontemplated that a nucleic acid fragment of almost any length may beemployed, with the total length preferably being limited by the ease ofpreparation and use in the intended recombinant nucleic acid protocol.In some cases, a nucleic acid sequence may encode a polypeptide sequencewith additional heterologous coding sequences, for example to allow forpurification of the polypeptide, transport, secretion,post-translational modification, or for therapeutic benefits such astargeting or efficacy. As discussed above, a tag or other heterologouspolypeptide may be added to the modified polypeptide-encoding sequence,wherein “heterologous” refers to a polypeptide that is not the same asthe modified polypeptide.

In certain embodiments, there are polynucleotide variants havingsubstantial identity to the sequences disclosed herein; those comprisingat least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or highersequence identity, including all values and ranges there between,compared to a polynucleotide sequence provided herein using the methodsdescribed herein (e.g., BLAST analysis using standard parameters). Incertain aspects, the isolated polynucleotide will comprise a nucleotidesequence encoding a polypeptide that has at least 90%, preferably 95%and above, identity to an amino acid sequence described herein, over theentire length of the sequence; or a nucleotide sequence complementary tosaid isolated polynucleotide.

A. Vectors

Polypeptides may be encoded by a nucleic acid molecule. The nucleic acidmolecule can be in the form of a nucleic acid vector. The term “vector”is used to refer to a carrier nucleic acid molecule into which aheterologous nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated and expressed. A nucleic acid sequencecan be “heterologous,” which means that it is in a context foreign tothe cell in which the vector is being introduced or to the nucleic acidin which is incorporated, which includes a sequence homologous to asequence in the cell or nucleic acid but in a position within the hostcell or nucleic acid where it is ordinarily not found. Vectors includeDNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses,and plant viruses), and artificial chromosomes (e.g., YACs). One ofskill in the art would be well equipped to construct a vector throughstandard recombinant techniques (for example Sambrook et al., 2001;Ausubel et al., 1996, both incorporated herein by reference). Vectorsmay be used in a host cell to produce an antibody that binds SpA.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. Expression vectors can contain avariety of “control sequences,” which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of an operablylinked coding sequence in a particular host organism. In addition tocontrol sequences that govern transcription and translation, vectors andexpression vectors may contain nucleic acid sequences that serve otherfunctions as well and are described herein.

A “promoter” is a control sequence. The promoter is typically a regionof a nucleic acid sequence at which initiation and rate of transcriptionare controlled. It may contain genetic elements at which regulatoryproteins and molecules may bind such as RNA polymerase and othertranscription factors. The phrases “operatively positioned,”“operatively linked,” “under control,” and “under transcriptionalcontrol” mean that a promoter is in a correct functional location and/ororientation in relation to a nucleic acid sequence to controltranscriptional initiation and expression of that sequence. A promotermay or may not be used in conjunction with an “enhancer,” which refersto a cis-acting regulatory sequence involved in the transcriptionalactivation of a nucleic acid sequence.

The particular promoter that is employed to control the expression of apeptide or protein encoding polynucleotide is not believed to becritical, so long as it is capable of expressing the polynucleotide in atargeted cell, preferably a bacterial cell. Where a human cell istargeted, it is preferable to position the polynucleotide coding regionadjacent to and under the control of a promoter that is capable of beingexpressed in a human cell. Generally speaking, such a promoter mightinclude either a bacterial, human or viral promoter.

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals.

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. (See Carbonelli et al., 1999, Levenson et al., 1998,and Cocea, 1997, incorporated herein by reference.)

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression. (SeeChandler et al., 1997, incorporated herein by reference.)

The vectors or constructs will generally comprise at least onetermination signal. A “termination signal” or “terminator” is comprisedof the DNA sequences involved in specific termination of an RNAtranscript by an RNA polymerase. Thus, in certain embodiments atermination signal that ends the production of an RNA transcript iscontemplated. A terminator may be necessary in vivo to achieve desirablemessage levels. In eukaryotic systems, the terminator region may alsocomprise specific DNA sequences that permit site-specific cleavage ofthe new transcript so as to expose a polyadenylation site. This signalsa specialized endogenous polymerase to add a stretch of about 200 Aresidues (polyA) to the 3′ end of the transcript. RNA molecules modifiedwith this polyA tail appear to more stable and are translated moreefficiently. Thus, in other embodiments involving eukaryotes, it ispreferred that that terminator comprises a signal for the cleavage ofthe RNA, and it is more preferred that the terminator signal promotespolyadenylation of the message.

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript.

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

B. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organism that is capable of replicating a vector orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors or viruses. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid, such as a recombinant protein-encoding sequence,is transferred or introduced into the host cell. A transformed cellincludes the primary subject cell and its progeny.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

C. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with an embodiment to produce nucleicacid sequences, or their cognate polypeptides, proteins and peptides.Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

In addition to the disclosed expression systems, other examples ofexpression systems include STRATAGENE®'s COMPLETE CONTROL™ InducibleMammalian Expression System, which involves a syntheticecdysone-inducible receptor, or its pET Expression System, an E. coliexpression system. Another example of an inducible expression system isavailable from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

D. Methods of Gene Transfer

Suitable methods for nucleic acid delivery to effect expression ofcompositions are believed to include virtually any method by which anucleic acid (e.g., DNA, including viral and nonviral vectors) can beintroduced into a cell, a tissue or an organism, as described herein oras would be known to one of ordinary skill in the art. Such methodsinclude, but are not limited to, direct delivery of DNA such as byinjection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448,5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, eachincorporated herein by reference), including microinjection (Harland andWeintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein byreference); by electroporation (U.S. Pat. No. 5,384,253, incorporatedherein by reference); by calcium phosphate precipitation (Graham and VanDer Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAEdextran followed by polyethylene glycol (Gopal, 1985); by direct sonicloading (Fechheimer et al., 1987); by liposome mediated transfection(Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wonget al., 1980; Kaneda et al., 1989; Kato et al., 1991); bymicroprojectile bombardment (PCT Application Nos. WO 94/09699 and95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783, 5,563,055, 5,550,318,5,538,877 and 5,538,880, and each incorporated herein by reference); byagitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat.Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); byAgrobacterium mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); or by PEG mediatedtransformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition mediated DNA uptake (Potrykus et al., 1985).Through the application of techniques such as these, organelle(s),cell(s), tissue(s) or organism(s) may be stably or transientlytransformed.

IV. METHODS OF TREATMENT

As discussed above, the compositions and methods of using thesecompositions can treat a subject (e.g., limiting bacterial load orabscess formation or persistence) having, suspected of having, or atrisk of developing an infection or related disease, particularly thoserelated to staphylococci. One use of the compositions is to preventnosocomial infections by inoculating a subject prior to hospitaltreatment.

As used herein the phrase “immune response” or its equivalent“immunological response” refers to a humoral (antibody mediated),cellular (mediated by antigen-specific T cells or their secretionproducts) or both humoral and cellular response directed against aprotein, peptide, or polypeptide of the invention in a recipientpatient. Treatment or therapy can be an active immune response inducedby administration of immunogen or a passive therapy effected byadministration of antibody, antibody containing material, or primedT-cells.

As used herein “passive immunity” refers to any immunity conferred upona subject by administration of immune effectors including cellularmediators or protein mediators (e.g., an polypeptide that binds to SpAprotein). An antibody composition may be used in passive immunizationfor the prevention or treatment of infection by organisms that carry theantigen recognized by the antibody. An antibody composition may includeantibodies or polypeptides comprising antibody CDR domains that bind toa variety of antigens that may in turn be associated with variousorganisms. The antibody component can be a polyclonal antiserum. Incertain aspects the antibody or antibodies are affinity purified from ananimal or second subject that has been challenged with an antigen(s).Alternatively, an antibody mixture may be used, which is a mixture ofmonoclonal and/or polyclonal antibodies to antigens present in the same,related, or different microbes or organisms, such as gram-positivebacteria, gram-negative bacteria, including but not limited tostaphylococcus bacteria.

Passive immunity may be imparted to a patient or subject byadministering to the patient immunoglobulins (Ig) or fragments thereofand/or other immune factors obtained from a donor or other non-patientsource having a known immunoreactivity. In other aspects, an antigeniccomposition can be administered to a subject who then acts as a sourceor donor for globulin, produced in response to challenge from thecomposition (“hyperimmune globulin”), that contains antibodies directedagainst Staphylococcus or other organism. A subject thus treated woulddonate plasma from which hyperimmune globulin would then be obtained,via conventional plasma-fractionation methodology, and administered toanother subject in order to impart resistance against or to treatstaphylococcus infection. Hyperimmune globulins are particularly usefulfor immune-compromised individuals, for individuals undergoing invasiveprocedures or where time does not permit the individual to produce theirown antibodies in response to vaccination. See U.S. Pat. Nos. 6,936,258,6,770,278, 6,756,361, 5,548,066, 5,512,282, 4,338,298, and 4,748,018,each of which is incorporated herein by reference in its entirety, forexemplary methods and compositions related to passive immunity.

For purposes of this specification and the accompanying claims the terms“epitope” and “antigenic determinant” are used interchangeably to referto a site on an antigen to which B and/or T cells respond or recognize.B-cell epitopes can be formed both from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of a protein.Epitopes formed from contiguous amino acids are typically retained onexposure to denaturing solvents whereas epitopes formed by tertiaryfolding are typically lost on treatment with denaturing solvents. Anepitope typically includes at least 3, and more usually, at least 5 or8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include those methodsdescribed in Epitope Mapping Protocols (1996). T cells recognizecontinuous epitopes of about nine amino acids for CD8 cells or about13-15 amino acids for CD4 cells. T cells that recognize the epitope canbe identified by in vitro assays that measure antigen-dependentproliferation, as determined by ³H-thymidine incorporation by primed Tcells in response to an epitope (Burke et al., 1994), byantigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al.,1996) or by cytokine secretion.

The presence of a cell-mediated immunological response can be determinedby proliferation assays (CD4 (+) T cells) or CTL (cytotoxic Tlymphocyte) assays. The relative contributions of humoral and cellularresponses to the protective or therapeutic effect of an immunogen can bedistinguished by separately isolating IgG and T-cells from an immunizedsyngeneic animal and measuring protective or therapeutic effect in asecond subject. As used herein and in the claims, the terms “antibody”or “immunoglobulin” are used interchangeably.

Optionally, an antibody or preferably an immunological portion of anantibody, can be chemically conjugated to, or expressed as, a fusionprotein with other proteins. For purposes of this specification and theaccompanying claims, all such fused proteins are included in thedefinition of antibodies or an immunological portion of an antibody.

In one embodiment a method includes treatment for a disease or conditioncaused by a staphylococcus pathogen. In certain aspects embodimentsinclude methods of treatment of staphylococcal infection, such ashospital acquired nosocomial infections. In some embodiments, thetreatment is administered in the presence of staphylococcal antigens.Furthermore, in some examples, treatment comprises administration ofother agents commonly used against bacterial infection, such as one ormore antibiotics.

The therapeutic compositions are administered in a manner compatiblewith the dosage formulation, and in such amount as will betherapeutically effective. The quantity to be administered depends onthe subject to be treated. Precise amounts of active ingredient requiredto be administered depend on the judgment of the practitioner. Suitableregimes for initial administration and boosters are also variable, butare typified by an initial administration followed by subsequentadministrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a polypeptide therapeutic are applicable.These are believed to include oral application on a solidphysiologically acceptable base or in a physiologically acceptabledispersion, parenterally, by injection and the like. The dosage of thecomposition will depend on the route of administration and will varyaccording to the size and health of the subject.

In certain instances, it will be desirable to have multipleadministrations of the composition, e.g., 2, 3, 4, 5, 6 or moreadministrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8,to 5, 6, 7, 8, 9, 10, 11, 12 twelve week intervals, including all rangesthere between.

A. Antibodies and Passive Immunization

Certain aspects are directed to methods of preparing an antibody for usein prevention or treatment of staphylococcal infection comprising thesteps of immunizing a recipient with a vaccine and isolating antibodyfrom the recipient, or producing a recombinant antibody. An antibodyprepared by these methods and used to treat or prevent a staphylococcalinfection is a further aspect. A pharmaceutical composition comprisingantibodies that specifically bind SpA and a pharmaceutically acceptablecarrier is a further aspect that could be used in the manufacture of amedicament for the treatment or prevention of staphylococcal disease. Amethod for treatment or prevention of staphylococcal infectioncomprising a step of administering to a patient an effective amount ofthe pharmaceutical preparation is a further aspect.

Inocula for polyclonal antibody production are typically prepared bydispersing the antigenic composition (e.g., a peptide or antigen orepitope of SpA or a consensus thereof) in a physiologically tolerablediluent such as saline or other adjuvants suitable for human use to forman aqueous composition. An immunostimulatory amount of inoculum isadministered to a mammal and the inoculated mammal is then maintainedfor a time sufficient for the antigenic composition to induce protectiveantibodies. The antibodies can be isolated to the extent desired by wellknown techniques such as affinity chromatography (Harlow and Lane,Antibodies: A Laboratory Manual 1988). Antibodies can include antiserumpreparations from a variety of commonly used animals e.g., goats,primates, donkeys, swine, horses, guinea pigs, rats or man. The animalsare bled and serum recovered.

An antibody can include whole antibodies, antibody fragments orsubfragments. Antibodies can be whole immunoglobulins of any class(e.g., IgG, IgM, IgA, IgD or IgE), chimeric antibodies, humanantibodies, humanized antibodies, or hybrid antibodies with dualspecificity to two or more antigens. They may also be fragments (e.g.,F(ab′)2, Fab′, Fab, Fv and the like including hybrid fragments). Anantibody also includes natural, synthetic or genetically engineeredproteins that act like an antibody by binding to specific antigens witha sufficient affinity.

A vaccine can be administered to a recipient who then acts as a sourceof antibodies, produced in response to challenge from the specificvaccine. A subject thus treated would donate plasma from which antibodywould be obtained via conventional plasma fractionation methodology. Theisolated antibody would be administered to the same or different subjectin order to impart resistance against or treat staphylococcal infection.Antibodies are particularly useful for treatment or prevention ofstaphylococcal disease in infants, immune compromised individuals orwhere treatment is required and there is no time for the individual toproduce a response to vaccination.

An additional aspect is a pharmaceutical composition comprising two ofmore antibodies or monoclonal antibodies (or fragments thereof;preferably human or humanized) reactive against at least twoconstituents of the immunogenic composition, which could be used totreat or prevent infection by Gram positive bacteria, preferablystaphylococci, more preferably S. aureus or S. epidermidis.

B. Combination Therapy

The compositions and related methods, particularly administration of anantibody that binds SpA or a peptide or consensus peptide thereof to apatient/subject, may also be used in combination with the administrationof traditional therapies. These include, but are not limited to, theadministration of antibiotics such as streptomycin, ciprofloxacin,doxycycline, gentamycin, chloramphenicol, trimethoprim,sulfamethoxazole, ampicillin, tetracycline or various combinations ofantibiotics.

In one aspect, it is contemplated that a therapy is used in conjunctionwith antibacterial treatment. Alternatively, the therapy may precede orfollow the other agent treatment by intervals ranging from minutes toweeks. In embodiments where the other agents and/or a proteins orpolynucleotides are administered separately, one would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the therapeutic composition would still be ableto exert an advantageously combined effect on the subject. In suchinstances, it is contemplated that one may administer both modalitieswithin about 12-24 h of each other and, more preferably, within about6-12 h of each other. In some situations, it may be desirable to extendthe time period for administration significantly, however, where severaldays (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8)lapse between the respective administrations.

Various combinations of therapy may be employed, for example antibiotictherapy is “A” and an antibody therapy that comprises an antibody thatbinds SpA or a peptide or consensus peptide thereof is “B”:

  A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the antibody compositions to a patient/subject willfollow general protocols for the administration of such compounds,taking into account the toxicity, if any, of the composition. It isexpected that the treatment cycles would be repeated as necessary. It isalso contemplated that various standard therapies, such as hydration,may be applied in combination with the described therapy.

C. General Pharmaceutical Compositions

In some embodiments, pharmaceutical compositions are administered to asubject. Different aspects may involve administering an effective amountof a composition to a subject. In some embodiments, an antibody thatbinds SpA or a peptide or consensus peptide thereof may be administeredto the patient to protect against or treat infection by one or morebacteria from the Staphylococcus genus. Alternatively, an expressionvector encoding one or more such antibodies or polypeptides or peptidesmay be given to a patient as a preventative treatment. Additionally,such compositions can be administered in combination with an antibiotic.Such compositions will generally be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium.

The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic, or other untoward reaction whenadministered to an animal or human. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredients, its use in immunogenic and therapeutic compositionsis contemplated. Supplementary active ingredients, such as otheranti-infective agents and vaccines, can also be incorporated into thecompositions.

The active compounds can be formulated for parenteral administration,e.g., formulated for injection via the intravenous, intramuscular,sub-cutaneous, or even intraperitoneal routes. Typically, suchcompositions can be prepared as either liquid solutions or suspensions;solid forms suitable for use to prepare solutions or suspensions uponthe addition of a liquid prior to injection can also be prepared; and,the preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The proteinaceous compositions may be formulated into a neutral or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

A pharmaceutical composition can include a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion, and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization or an equivalent procedure. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques, which yield a powder of the active ingredient,plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Administration of the compositions will typically be via any commonroute. This includes, but is not limited to oral, nasal, or buccaladministration. Alternatively, administration may be by orthotopic,intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal,or intravenous injection. In certain embodiments, a vaccine compositionmay be inhaled (e.g., U.S. Pat. No. 6,651,655, which is specificallyincorporated by reference). Such compositions would normally beadministered as pharmaceutically acceptable compositions that includephysiologically acceptable carriers, buffers or other excipients.

An effective amount of therapeutic or prophylactic composition isdetermined based on the intended goal. The term “unit dose” or “dosage”refers to physically discrete units suitable for use in a subject, eachunit containing a predetermined quantity of the composition calculatedto produce the desired responses discussed above in association with itsadministration, i.e., the appropriate route and regimen. The quantity tobe administered, both according to number of treatments and unit dose,depends on the protection desired.

Precise amounts of the composition also depend on the judgment of thepractitioner and are peculiar to each individual. Factors affecting doseinclude physical and clinical state of the subject, route ofadministration, intended goal of treatment (alleviation of symptomsversus cure), and potency, stability, and toxicity of the particularcomposition.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeutically orprophylactically effective. The formulations are easily administered ina variety of dosage forms, such as the type of injectable solutionsdescribed above.

V. EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments and are not meant to limit the present invention in anyfashion. One skilled in the art will appreciate readily that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those objects, ends and advantagesinherent herein. The present examples, along with the methods describedherein are presently representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1

Monoclonal Antibodies to Staphylococcus Aureus Protein A

SpA_(KKAA)-mAbs Protect Mice Against Staphylococcal Disease.

BALB/c mice were immunized with purified SpA_(KKAA) using aprime-booster regimen and antigen specific IgG responses were quantifiedby ELISA. Animals were euthanized and their splenocytes fused withmyeloma cells. The resulting hybridomas were screened for the productionof antigen-specific mAbs. Initially, protein A-specific mAbs werescreened using the functional assays as well as the murine infectionmodel (Table 1). After the initial screen, we selected three mAbs (5A10,3F6, and 3D11) for further characterization as these antibodiesdisplayed the best immune protection in each isotype group (Table 1).BALB/c Mice were immunized with affinity purified mAbs (5 mg·kg⁻¹ bodyweight) and challenged by injecting 1×10⁷ CFU S. aureus Newman, amethicillin-sensitive clinical isolate (MSSA) (Baba et al., 2007), intothe periorbital venous sinus of the right eye. The ability ofstaphylococci to seed abscesses in renal tissues was examined byhistopathology four days after challenge (Table 1). In homogenized renaltissues of control mice (immunized with 5 mg·kg⁻¹ isotype control mAbs),an average staphylococcal load of 5.02 log₁₀ CFU·g⁻¹ (IgG₁), 4.64 log₁₀CFU·g⁻¹² (IgG_(2a)) and 5.24 log₁₀ CFU·g⁻¹ (IgG_(2b)) was recovered(Table 1). Compared to isotype mAb-treated controls, animals thatreceived protein A specific mAbs displayed a reduction in staphylococcalload [2.80 log₁₀ CFU·g⁻¹ (5A10), 2.28 log₁₀ CFU·g⁻¹ (3F6), and 2.72log₁₀ CFU·g⁻¹ (3D11)] as well as abscess formation (Table 1). Of note,not all SpA_(KKAA)-mAbs generated protection against staphylococcaldisease (Table 1) even though these antibodies bound with appreciableaffinity to their antigen (see for example 3A6 and 6D11 in Table 3).

TABLE 1 Passive immunization of mice with monoclonal antibodies againstSpA_(KKAA) Staphylococcal load and abscess formation in renal tissue^(b)log₁₀CFU ^(c)P ^(d)Reduc- ^(e)Number of ^(c)P ^(a)Antibody g⁻¹ valuetion abscesses value IgG₁ Mock 5.02 ± 0.66 — — 2.00 ± 0.94 — 5A10 2.22 ±0.22 0.0019 2.80 0.00 ± 0.00 0.0350 8E2 3.01 ± 0.37 0.0629 2.01 0.20 ±0.20 0.1117 3A6 3.98 ± 0.47 0.3068 1.04 0.50 ± 0.50 0.1497 7E2 5.01 ±0.64 0.9396 0.01 2.00 ± 0.99 0.7461 IgG_(2a) Mock 4.64 ± 0.49 — — 3.70 ±1.40 — 3F6 2.36 ± 0.36 0.0010 2.28 0.60 ± 0.50 0.0239 1F10 3.05 ± 0.460.0299 1.59 0.70 ± 0.40 0.0812 6D11 3.88 ± 0.75 0.1967 0.76 0.90 ± 0.350.1793 IgG_(2b) Mock 5.24 ± 0.51 — — 3.00 ± 0.67 — 3D11 2.52 ± 0.400.0010 2.72 0.56 ± 0.28 0.0068 5A11 3.26 ± 0.55 0.0171 1.98 0.80 ± 0.550.0286 1B10 3.31 ± 0.34 0.0113 1.93 0.50 ± 0.50 0.0070 4C1 3.38 ± 0.500.0228 1.86 0.10 ± 0.10 0.0016 2F2 3.49 ± 0.70 0.0232 1.75 0.40 ± 0.270.0424 8D4 3.83 ± 0.63 0.1198 1.41 0.80 ± 0.51 0.0283 7D11 4.23 ± 0.550.2729 1.01 0.90 ± 0.55 0.0424 2C3 4.24 ± 0.61 0.1733 1.00 1.40 ± 0.600.1623 4C5 4.35 ± 0.53 0.2410 0.89 1.90 ± 0.84 0.3270 6B2 4.42 ± 0.620.4055 0.82 2.20 ± 1.00 0.3553 4D5 4.96 ± 0.58 0.7912 0.28 3.80 ± 1.260.7884 2B8 5.00 ± 0.66 0.8534 0.24 4.60 ± 2.89 0.6184 1H7 5.59 ± 0.430.5675 −0.35  2.89 ± 0.73 0.9008 ^(a)Affinity purified antibodies wereinjected into the peritoneal cavity of BALB/c mice at a concentration of5 mg kg⁻¹ four hours prior to intravenous challenge with 1 × 10⁷ CPU S.aureus Newman. ^(b)Means (±SEM) of staphylococcal load calculated aslog₁₀ CFU g⁻¹ in homogenized renal tissues 4 days following infection incohorts often BALB/c mice per immunization. A representative of threeindependent and reproducible animal experiments is shown.^(c)Statistical significance was calculated with the unpaired two-tailedMann-Whitney test and P-values recorded. ^(d)Reduction in bacterial loadcalculated as log₁₀ CFU g⁻¹. ^(e)Histopathology of hematoxylin-eosinstained, thin sectioned kidneys from ten animals; the number ofabscesses per kidney was recorded and averaged for the final mean(±SEM).

SpA_(KKAA)-mAbs Protect Mice Against MRSA Challenge.

Cohorts of BALB/c mice were immunized with mAbs 5A10, 3F6, 3D11 (5mg·kg⁻¹) or a combination of all three mAbs (15 mg·kg⁻¹) and challengedwith strain MW2, a highly virulent community-acquired, MRSA isolate(Baba et al., 2002). Compared to isotype mAb-treated controls, animalsthat received any one of the three mAbs (5A10, 3F6, 3D11) harbored areduced bacterial load and fewer staphylococcal abscesses in renaltissues (Table 2). Animals that had been immunized with a mixture of allthree mAbs (15 mg·kg⁻¹) displayed an even greater reduction instaphylococcal load (2.03 log₁₀ CFU·g⁻¹ reduction; P<0.0002) and inabscess formation (vaccine vs. mock, P<0.0004). It is likely thatenhanced protection is due to administration of increased concentrationof mAbs (15 mg·kg⁻¹ vs. 5 mg·kg⁻¹). We arrived at this hypothesisbecause the three antibodies, although recognizing similar structuralfeatures, do not appear to occupy identical binding sites on SpA (videinfra). Further, increasing the concentration of only one of the threemAbs (3F6) caused the same effect: increased protection againststaphylococcal disease (vide infra).

In addition to providing immediate protection against staphylococcalchallenge, SpA_(KKAA)-specific mAbs may also neutralize the B-cellsuperantigen activity of SpA (Goodyear and Silverman, 2003), therebyenabling infected hosts to generate antibody responses against manydifferent staphylococcal antigens (Kim et al., 2010a). To examine thispossibility, BALB/c mice were passively immunized with mAb 3F6 or itsIgG_(2a) isotype control (20 mg·kg⁻¹) prior to intravenous challengewith S. aureus MW2. Fifteen days after challenge, animals wereeuthanized and staphylococcal load in organ tissue examined (FIG. 1A).Mice that had been immunized with mAb 3F6 harbored a reducedstaphylococcal load (4.77 log₁₀ CFU·g⁻¹ reduction, P=0.0013) as well asa reduced number of abscesses [from 10.14 (±2.08) (IgG_(2a)) to 3.00(±1.00) (3F6), P=0.0065; FIG. 1A]. Blood samples withdrawn 15 dayspost-challenge were examined for serum IgG reactive against fourteenstaphylococcal antigens under consideration as protective antigens forvaccine development: Coa, ClfA, ClfB, EsxA, EsxB, FnBPA, FnBPB, Hla,IsdA, IsdB, LukD, SdrD, SpA_(KKAA) and vWbp (DeDent et al., 2012). Asobserved previously with animals that had been actively vaccinated withSpA_(KKAA), mice that had been immunized with mAb 3F6 developed higherserum IgG titers against several different staphylococcal antigens (FIG.1B) (Kim et al., 2010a). In particular, IgG levels against Coa, ClfA,EsxA, EsxB, FnBPB, Hla, IsdA, LukD, SdrD and vWbp were increased inserum samples of mAb 3F6-immunized animals as compared to the controlcohort. Nevertheless, serum IgG against IsdB, the staphylococcalhemoglobin hemophore (Mazmanian et al., 2003), was not increased (FIG.1B). The IgG titer against SpA_(KKAA) was sustained over fifteen daysfollowing passive transfer of mAb 3F6 (FIG. 1B).

During staphylococcal infection, recognition of soluble SpA by mAb 3F6is expected to form immune complexes (IC) that are then phagocytosed byimmune cells. Phagocytosed SpA is then processed by proteolytic enzymesin the phagolysosome and peptide fragments are presented to T and Bcells to produce polyclonal antibodies. As a confirmatory test, cohortsof animals received a mixture of affinity purified recombinant protein Avariants [SpA, SpA_(KK), SpA_(AA), SpA_(KKAA), and mock (PBS)] in thepresence of mAb 3F6 or its isotype control at day 0 and 11. At day 21,animals were euthanized and their ability to elicit different classes ofSpA-specific antibody was measured by ELISA. All animals failed togenerate SpA-specific antibody responses without mAb treatment (FIG. 6).In addition, animals that received B cell superantigens (SpA andSpA_(KK); vide infra) failed to generate SpA-specific IgG₁ and IgG_(2a)antibodies even in the presence of mAb 3F6 (FIG. 6). However, micetreated with SpA variants lacking B cell superantigen activity (SpA_(AA)and SpA_(KKAA); vide infra) were able to generate a significant amountof IgG₁ (FIG. 6). Although the estimated amount of soluble protein Aduring infection (5˜10 ng per 10⁷ CFU) is well below the dose ofaffinity purified protein A injected in these experiments into animals,the data in FIG. 6 suggest a potential role of SpA-specific T/B cells inneutralizing B cell superantigen activity. Taken together, the inventorspresume that active vaccination with SpA_(KKAA) (Kim et al., 2010a), butnot passive immunization of S. aureus infected mice with neutralizingmAbs (vide infra) can raise a significant level of protein A-specificantibodies.

TABLE 2 Immunization with SpA_(KKAA)-mAbs protects mice against MRSAchallenge Staphylococcal load and abscess formation in renal tissue^(b)log₁₀CFU ^(c)P Reduc- ^(e)Number of ^(c)P ^(a)Antibody g⁻¹ valuetion abscesses value IgG₁ Mock 7.42 ± 0.20 — — 22.3 ± 6.3 — 5A10 6.00 ±0.21 0.0009 1.42 10.2 ± 2.5 0.0482 IgG_(2a) Mock 7.15 ± 0.18 — — 11.8 ±2.0 — 3F6 5.80 ± 0.21 0.0009 1.35  6.4 ± 0.7 0.0323 IgG_(2b) Mock 7.13 ±0.11 — — 14.0 ± 1.8 — 3D11 5.81 ± 0.25 0.0006 1.32  7.7 ± 1.9 0.0489IgG₁ + IgG_(2a) + IgG_(2b) Mock 7.75 ± 0.06 — — 17.4 ± 1.7 — 5A10/3F6/5.72 ± 0.12 0.0002 2.03  6.7 ± 0.6 0.0004 3D11 ^(a)Affinity purifiedantibodies were injected into the peritoneal cavity of BALB/c mice at aconcentration of individual antibody at 5 mg · kg⁻¹ or combinations ofthree monoclonal antibodies at 15 mg · kg⁻¹ twenty four hours prior tointravenous challenge with 1 × 10⁷ CFU S. aureus MW2. ^(b)Means (±SEM)of staphylococcal load calculated as log₁₀CFU · g⁻¹ in homogenized renaltissues 4 days following infection in cohorts of ten BALB/c mice perimmunization with limit of detection at 1.99 log₁₀CFU · g⁻¹. Arepresentative of two independent and reproducible animal experiments isshown. ^(c)Statistical significance was calculated with the two-tailedMann-Whitney test and P-values recorded. ^(d)Reduction in bacterial loadcalculated as log₁₀CFU · g⁻¹. ^(e)Histopathology of hematoxylin-eosinstained, thin sectioned kidneys from ten animals; the number ofabscesses per kidney was recorded and averaged for the final mean(±SEM).

mAb Spa27 does not Recognize SpA_(KKAA) and Fails to Elicit ProtectiveImmunity in Mice.

Spa27 is a commercially available protein A-specific monoclonal antibody(Sigma) that has been used over the past two decades for the detectionof staphylococcal protein A (Perry et al., 2002). The Spa27 hybridomawas generated from mice that had been immunized with wild-typestaphylococcal protein A purified from S. aureus strain Cowan I(Sjoquist et al., 1972). Previous work demonstrated that wild-typeprotein A triggers the clonal expansion and collapse of B cellpopulations (Forsgren et al., 1976, Goodyear et al., 2003), therebyablating protein A-specific immune response in mice (Goodyear et al.,2004), and that wild-type protein A encompasses binding sites for bothFcγ and Fab V_(H)3 (Graille et al., 2000, Stahlenheim et al., 1970). Wetherefore wondered whether Spa27 recognizes wild-type protein A as anantigen. To address this question, we used ELISA with purifiedrecombinant protein A (SpA) or its variants that lack either the abilityto specifically bind Fcγ (SpA_(KK)), the Fab domain of V_(H)3 (SpA_(AA))or both (SpA_(KKAA)) (FIG. 7). The data revealed strong binding of Spa27to wild-type SpA and SpA_(KK), but not to SpA_(AA) or SpA_(KKAA) (FIG.9B). Spa27 is a mouse IgG₁ isotype antibody, which explains itsinability to bind protein A via Fcγ (Kronvall et al., 1970). The weakassociation between Spa27 and SpA_(AA) or SpA_(KKAA) could be due to theseemingly remote possibility that SpA27 requires residues D36/D37 ineach of the five IgBDs for antigen recognition or, more likely to us,that Spa27 binds SpA via its Fab domain, assuming the antibody belongsto the V_(H)3 or a related class of antibody.

We examined the biological function of Spa27 by injecting for pairwisecomparison mAbs 3F6 or Spa27 (5 mg·kg⁻¹) into the peritoneal cavity ofBALB/c mice. These animals were then challenged with S. aureus USA300(LAC), the highly virulent community-acquired MRSA strain epidemic inthe United States (Diep et al., 2006). At 4 days post challenge, animalswere euthanized and the bacterial load in the kidneys of infectedanimals were determined (FIG. 9B). Compared to mock (PBS) treatment,animals that received mAb 3F6 harbored a reduced bacterial load (1.38log₁₀ CFU·g⁻¹ reduction, P=0.0011). In contrast to the protectiveimmunity elicited by 3F6, mAb Spa27 failed to reduce the bacterial loadin kidneys of infected animals (0.20 log₁₀ CFU·g⁻¹ reduction, P=0.2111).These data reveal that mAb Spa27 does not provide protection againststaphylococcal disease. Further, the experiments with Spa27 illustratethat immunization of mice with wild-type protein A may not elicitmonoclonal antibodies that can neutralize the immune-modulatoryattributes of protein A by binding this molecule as an antigen.

Recognition of SpA_(KKAA) by mAbs.

Microtiter dishes were coated with SpA_(KKAA) and ELISA was used todetermine the affinity constant (K_(a)=[mAb·Ag]/[mAAb]×[Ag]) of purifiedmAbs. mAb 3F6 displayed the highest affinity (K_(a) 22.97×10⁹ M⁻¹)followed by mAb 5A10 (K_(a) 8.47×10⁹ M⁻¹) and mAb 3D11 (K_(a) 3.93×10⁹M⁻¹, Table 3). Each of the five IgBDs alone (E_(KKAA), D_(KKAA),A_(KKAA), B_(KKAA) and C_(KKAA)) or peptides encompassing helix 1, 2 or3 as well as helices 1+2 and 2+3 of the IgBD E_(KKAA) domain wereexamined for antibody binding (Table 3). mAbs 5A10 and 3F6 bound allfive IgBDs with the same affinity as SpA_(KKAA). mAb 5A10 did not bindto the helical peptides, whereas mAb 3F6 displayed weak affinity for thehelix 1+2 peptide. mAb 3D11 bound to B_(KKAA) and C_(KKAA) and weakly toA_(KKAA), but not to E_(KKAA) and D_(KKAA). In sum, SpA_(KKAA)-mAbs thatafforded the highest levels of protection against staphylococcal diseasein mice bound some or all of the five IgBDs, but not the peptidesencompassing only one or two of three helices of IgBDs. These datasuggest that protective mAbs recognize conformational epitopes of thetriple-helical bundle for each IgBD.

To examine whether the avidities of mAbs play a significant role inimmune protection, ELISA was performed in the presence of increasingconcentrations of the chaotropic reagent ammonium thiocyanate (FIG. 2).The measured avidity of mAb 3F6 was significantly higher than that ofmAb 5A10 and 3D11 (FIG. 2). Of note, 3D11 displayed relatively lowavidity, which may be due to its specific interaction with only two ofthe five IgBDs (FIG. 2 and Table 3). From this we conclude that theavidities of mAbs may not be a major determinant of their immuneprotection in mice.

United States Patent Application Publications US 2008/0118937 and US2010/0047252 describe a murine hybridoma cell line that was derived froma mouse following immunization with wild-type protein A. Thecorresponding antibody, mAb 358A76.1.1, was reported to associate withwild-type protein A; however, the molecular nature of this associationhas not yet been revealed. To further explore the nature of B cellresponses to wild-type protein A, mAb 358A76.1.1 was isolated andexamined for its functional attributes. Unlike SpAKKAA-mAbs, the358A76.1 antibody bound only to the E domain of SpA, but not any of thefour other IgBDs (D, A, B and C). Furthermore, mAb 358A76.1 neitherneutralized protein A nor promoted opsonophagocytic killing ofstaphylococci, and passive transfer of mAb 358A76.1 did not protect miceagainst S. aureus disease.

Monoclonal Antibody 358A76.1 Weakly Binds to Only the E Domain of SpA.

To determine the affinity constant (K_(a)=[mAb·Ag]/[mAb]×[Ag]) of mAb358A76.1 for binding to protein A, microtiter plates were coated witheither SpA_(KKAA), individual IgBDs (E_(KKAA), D_(KKAA), A_(KKAA),B_(KKAA) or C_(KKAA)), as well as synthetic peptides encompassingindividual helixes (H1, H2, and H3) or two helices (H1+2 and H2+3) ofthe E_(KKAA) triple helical bundle. mAb 3F6 is a SpA_(KKAA)-derivedmouse monoclonal antibody with high affinity for full-length SpA_(KKAA)(K_(a) 22.97×10⁹ M⁻¹) and each of the five IgBDs (K_(a) 12.41-27.46×10⁹M⁻¹). Compared to mAb 3F6, mAb 358A76.1 displayed much weaker affinityfor SpA_(KKAA) (K_(a) 1.00×10⁹ M⁻¹, FIG. 10A-B). Of note, mAb 358A76.1bound only to E_(KKAA) (K_(a) 0.21×10⁹ M⁻¹) but not to any of the otherfour IgBDs (D_(KKAA), A_(KKAA), B_(KKAA) or C_(KKAA), Table 6).Furthermore, mAb 358A76.1 did not recognize any of the syntheticpeptides encompassing one or two helices of the E_(KKAA) IgBD (H1, H2,H3, H1+2, and H2+3). In contrast, mAb 3F6 displayed weak affinity forhelix 1+2 peptide (Table 6). An alignment of the amino acid sequences ofall five IgBDs revealed that the E domain is the most dissimilar domain(Sjodahl, 1977). Nevertheless, the E domain, just like the other fourIgBDs, associates with human and animal immunoglobulin and thesignificance of its dissimilarity has not yet been appreciated (Moks etal., 1986) (FIG. 10C-D). It is possible that mAb 358A76.1 specificallybinds a conformational epitope involving the non-conserved amino acidsof helix 1 and 3 of the E domain (FIG. 10C-D).

To examine whether mAbs 3F6 and 358A76.1 share epitope binding sites onSpA_(KKAA) (E_(KKAA)), we performed competitive ELISA using increasingconcentrations of IgG_(2a) isotype control, mAb 358A76.1 or mAb 3F6. At30 μg·ml⁻¹ concentration, isotype control antibody (IgG_(2a)) did notinterfere with the binding of HRP-conjugated mAb 358A76.1 or mAb 3F6 toSpA_(KKAA) (FIG. 10E). As expected, mAb 358A76.1 competed withHRP-conjugated mAb 358A76.1 for binding to SpA_(KKAA), however it didnot interfere with the binding of HRP-conjugated mAb 3F6 (FIG. 10E). Ofnote, mAb 3F6 was able to completely block the association ofHRP-conjugated mAb 358A76.1 to SpA_(KKAA) (96.4% inhibition, FIG. 10E),whereas mAb 358A76.1 generated only 88.0% inhibition (mAb 3F6 vs. mAb358A76.1, P=0.0007).

Monoclonal Antibody 358A76.1 does not Elicit Protective Immunity inMice.

Cohorts of BALB/c mice were injected into the intraperitoneal cavitywith 5 mg·kg⁻¹ mAb 358A76.1 or mAb 3F6. Passively immunized animals werechallenged with S. aureus USA300 (LAC), the highly virulentcommunity-associated MRSA strain that is epidemic in the United States(Diep et al., 2006, Kennedy et al., 2008). Compared to IgG2a isotypemAb-treated controls, animals that received mAb 3F6 harbored a reducedbacterial load in renal tissues (1.26 log₁₀ CFU·g⁻¹ reduction; P=0.0021,FIG. 11A). Interestingly, animals that received mAb 358A76.1 displayedonly a small reduction in bacterial load, which failed to achievestatistical significance (0.42 log₁₀ CFU·g⁻¹ reduction; P=0.0948, FIG.11A). Compared to mAb 358A76.1, passive transfer of mAb 3F6 generatedincreased protection against CA-MRSA strain USA300 in immunized mice(0.84 log₁₀ CFU·g⁻¹ reduction; P=0.0011, FIG. 11A).

Opsonophagocytic killing of invasive microbes is a key defense strategyof infected hosts and also represents a correlate of protective immunityfor many different bacterial vaccines (Robbins et al., 1987, Robbins etal., 1990). Using an assay of opsonophagocytic killing in fresh mouseblood, we asked whether mAb 358A76.1 can promote opsonophagocytickilling of MRSA strain USA300. Briefly, anti-coagulated blood obtainedfrom naïve 6 week old BALB/c mice was incubated with S. aureus USA300 inthe presence or absence of 10 μg·ml⁻¹ mAb 358A76.1, mAb 3F6 or mAb IgG2aisotype control. Blood samples were lysed, plated on agar medium andstaphylococcal load enumerated. In contrast to mAb 3F6, which reducedthe staphylococcal load in blood by 49%, mAbs 358A76.1 and IgG2a-controlfailed to activate opsonophagocytic killing of staphylococci (mAb 3F6vs. PBS, P<0.0001; mAb 3F6 vs. 358A76.1, P=0.0007; FIG. 11B).

During infection, protein A captures and decorates the surface ofstaphylococci with immunoglobulin. By associating with the Fcγ domain ofimmunoglobulin, SpA blocks complement activation, engagement Fcreceptors and opsonization of staphylococci by phagocytes. Furthermore,SpA molecules that are released from the bacterial surface crosslinkV_(H)3-type B cell receptors to activate lymphocytes, eventuallytriggering their apoptotic demise and interfering with the developmentof adaptive immune responses against staphylococci. We used ELISA todetermine whether mAb 358A76.1 can neutralize the immunoglobulin bindingactivities of protein A. As controls, at 6 μg·ml⁻¹ and 30 μg·ml⁻¹ mAb3F6 neutralized the ability of protein A to bind human IgG binding,whereas the IgG2a isotype control mAb did not (FIG. 11C). Furthermore,at a concentration of 6 μg·ml⁻¹ or 30 μg·ml⁻¹ mAb 358A76.1 did not blockthe association of the human IgG to protein A (FIG. 11C).

TABLE 3 Association constants of mAb binding to SpA_(KKAA) and itsfragments Association constant (nM⁻¹) SpA IgG binding domains Helixmotif of SpA-E ^(a)Antibody SpA_(KKAA) E D A B C H1 H2 H3 H1 + 2 H-2 + 3IgG₁ 5A10 8.47 9.40 8.19 8.08 7.03 10.12 < < < < < 8E2 1.56 1.40 1.511.52 1.14 1.26 < < < 0.29 < 3A6 1.37 1.38 < 2.05 0.64 0.04 0.06 < 0.010.44 < 7E2 0.31 0.29 0.30 0.36 0.32 0.28 < < < < < IgG_(2a) 3F6 22.9717.69 12.41 20.15 27.46 26.46 < 0.01 < 0.41 0.01 1F10 2.46 2.21 1.802.12 2.85 2.70 < < < 0.63 < 6D11 5.37 4.34 2.42 2.23 3.34 4.75 0.27 0.01< 5.22 0.00 IgG_(2b) 3D11 3.93 < < 0.87 3.92 3.60 0.02 < < < < 5A11 8.755.10 5.75 6.61 5.03 6.04 < < < 0.02 < 1B10 4.31 4.35 2.78 2.74 2.30 4.21< < < < 0.01 4C1 4.68 2.38 2.56 3.02 3.21 2.99 0.07 0.01 0.01 1.95 0.042F2 1.90 1.72 1.76 1.37 1.13 1.8 < < < < < 8D4 10.47 7.65 9.85 11.940.07 < 3.20 < < 4.88 < 7D11 5.46 3.14 3.51 4.15 4.62 6.02 < < < < < 2C36.84 5.35 3.41 4.25 3.90 6.33 < < < < < 4C5 4.42 < 1.76 4.57 1.8 2.11 << < < < 6B2 4.47 3.2 2.52 4.19 4.55 4.23 0.05 0.23 < 4.54 < 4D5 6.17 < <5.30 4.89 5.24 < < < < < 2B8 4.79 2.33 2.25 3.05 3.68 3.06 < 0.23 < 3.37< 1H7 2.86 2.42 2.17 2.37 2.57 4.43 < < < < < ^(a)Affinity purifiedantibodies (1 mg ml⁻¹) were serially diluted across the ELISA platecoated with cognate antigens (100 nM) to measure the associationconstant by Prism (GraphPad Software, Inc.).

TABLE 6  Association constants for the binding of mAbs 358A76 and 3F6 to SpA_(KKAA) and its fragmentsAssociation constant (×10⁹ M⁻¹) for antigen or antigen fragment ^(a)mAbIgG binding domains Segments of the E_(KKAA) of protein Atriple-helical bundle IgG_(2a) SpAKKAA EKKAA DKKAA AKKAA BKKAA CKKAA H1H2 H3 H1 + 2 H2 + 3 358A76  1.00  0.21 < < < < < < < < < 3F6 22.97 17.6912.41 20.15 27.46 26.46 < 0.01 < 0.41 0.01 ^(a)Affinity purifiedantibodies (100 μg·ml¹) were serially diluted across ELISA plates coatedwith antigens (20 nM for SpA_(KKAA) and 100 nM for IgG binding domains)to calculate the association constant using Prism ® (GraphPad Software,Inc.). To study the binding of antibodies to protein A antigen, we usedthe SpA. variant (residues 1-291 of mature SpA harboring six N-terminalhistidyl residues) with four amino acid substitutions in each of thefive immunoglobulin binding domains (IgBD) of protein A [E (residues1-56), D (residues 57-117), A (residues 118-B (residues 176-233) and C(residues 234-291)[. In each IgBD, the glutamines at position 9 and 10(amino acid residues from IgBD-E) were replaced with lysine (Q⁹K, Q¹⁰K)and aspartic acids 36 and 37 were substituted with alanine (D³⁶A, 0³⁷A).The same substitutions were introduced into proteins spanning individualIgBDs: EKKAA, D_(KKAA), A_(KKAA), B_(KKAA) and C_(KKAA) (all expressedand purified with an N-terminal six histidyl tag). SpA. and individualIgBDs were purified by affinity chromatography from E. coli extracts.Peptides H1, H2, H3, H1 + 2, and H2 + 3 were synthesized on a peptidesynthesizer and purified via HPLC. The peptides encompass helices 1 (H1:NH₂-AQHDEAKKNAPYQVLNMPNLNA-COOH), 2 (H2:NH₂-NMPNLNADQRNGFIQSLKAAPSQ-COOH), 3 (H3:NH₂-AAPSQSANVLGEAQKLNDSQAPK-COOH), 1 + 2 (H1 + 2:NH₂-AQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQ-COOH) or 2 + 3 (H2 + 3:NH₂-NMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPK-COOH) of the triplehelical bundle of the E_(KKAA) IgBD (residues 1-56 of SpA_(KKAA)NH₂-AQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPK-COOH).^(b)The symbol < signifies measurements that were too low to permit thedetermination of the association constant.

mAb 3F6 Binds Sbi.

Sbi, a secreted protein of S. aureus, is comprised of five distinctdomains (Zhang et al., 1998). Two N-terminal domains (1 and 2) arehomologous to the IgBDs of SpA (Zhang et al., 1999). Domains 3 and 4associate with complement components C3 and factor H and the C-terminaldomain has been proposed to retain some secreted Sbi molecules in thestaphylococcal envelope by binding to lipoteichoic acids (Burman et al.,2008, Smith et al., 2012). Domains 1 and 2 bind to the Fcγ portion ofimmunoglobulins (Atkins et al., 2008); this activity, in concert withthe C3 and factor H binding attributes of domains 3 and 4, promotes thefutile consumption of fluid complement components (Haupt et al., 2008).Sbi does not seem to exert B cell superantigen activity, as its twoIgBDs (domains 1 and 2) lack the canonical two aspartic acid residues atposition 36 and 37 (Graille et al., 2000, Lim et al 2011). His-Sbi₁₋₄, arecombinant protein encompassing both IgBDs and the complement bindingdomains, retained human IgG in an affinity chromatography experiment.His-Sbi_(1-4/KKAA) is a variant with lysine (K) substitutions ofconserved glutamine residues (Q^(51,52) and Q^(103,104)) in domains 1and 2, i.e. the predicted Fcγ binding sites of the Sbi IgBDs, andalanine (A) substitutions of arginine (R²³¹) and aspartic acid (D²³⁸)residues of the complement binding domain (Haupt et al., 2008).His-Sbi_(1-4/KKAA) did not retain human IgG during affinitychromatography. When examined by ELISA, His-Sbi₁₋₄ bound to mouse aswell as human IgG and to both the Fc and Fab domains of human IgG,whereas His-Sbi_(1-4/KKAA) did not. mAbs 5A10 and 3D11 did not bind toHis-Sbi_(1-4/KKAA), however 3F6 bound to the protein. Thus, mAb 3F6 mayneutralize Sbi or remove secreted Sbi from circulation, therebypreventing the consumption of complement factor C3 by staphylococci.

Binding site competition experiments with SpA_(KKAA)-mAbs. ELISA studiesrevealed that the three mAbs 5A10, 3F6 and 3D11 bound with similaraffinities to wild-type SpA (FIG. 3A). Compared to 5A10, the IgG₁control antibodies displayed little affinity for SpA. Further, theaffinity of the IgG_(2b) control antibody was reduced compared to thatof mAb 3D11. Compared to 3F6, the IgG_(2a) control antibody bound SpAwith slightly reduced affinity. In a competitive ELISA assay withhorseradish peroxidase conjugated mAbs (5A10-HRP, 3F6-HRP and 3D11-HRP),isotype control antibodies did not interfere with the binding ofHRP-conjugated mAbs to SpA (FIG. 3B). The addition of equimolar amountsof each mAb reduced the binding of the corresponding HRP-conjugate (FIG.3B). mAb 3D11 did not prevent the association of HRP-5A10 or HRP-3F6with SpA, however mAbs 5A10 and 3F6 interfered with HRP-3D11 binding toSpA. mAb 3F6 caused some reduction in the binding of HRP-5A10 to SpA(FIG. 3B). Finally, mAb 5A10 was a weak competitor for the binding of3F6-HRP to SpA (FIG. 3B). These data suggest that the binding sites forthe three mAbs on the surface of the triple-helical bundles of SpA maybe in close proximity to one another or even partially overlap (Table3).

SpA_(KKAA)-mAbs Prevent the Association of Immunoglobulin with proteinA.

Mouse antibodies of clan V_(H)3 related families (e.g. 7183, J606 and5107) bind SpA via their Fab portion, whereas those of other VH families(J558, Q52, Sm7, VH10, VH11 and VH12) do not (Cary et al., 1999). Theamino acid sequence of the complementarity determining region (CDR) ofSpA_(KKAA)-specific mAbs was determined by sequencing cDNA derived fromhybridoma transcripts. The data showed that mAb 5A10 belongs to the clanVH3 7183 family; its Fab domain likely displays affinity for SpA (Table4). mAbs 3F6 and 3D11 are members of the VH10 and J558 families,respectively (Table 4); Fab domains of these antibody families are notknown to associate with SpA.

TABLE 4  Amino acid sequences of CDR regions of monoclonal antibodiesAmino acid sequencing data of protein A specific monoclonal antibodies^(a)mAb MouseVH family CDR1 CDR2 CDR3 SA10 7183 ...SSVSY... ...DTS......QQWSSYPPT... 3F6 VH10 ...ESVEYSGASL... ...AAS... ...QQSRKVPST... 3D111558 ...SSVSY... ...EIS... ...QQWSYPFT... ^(a)Amplified PCR productsfrom cDNA which was synthesized from total RNA extracted from hybridomacells were sequenced and analyzed using IMGT Vquest.

Wild-type SpA and its variants SpA_(KKAA), SpA_(KK) and SpA_(AA) werepurified and used for ELISA binding studies with human IgG. As expected,SpA bound to IgG or its Fcγ and F(ab)₂ fragments, whereas SpA_(KKAA) didnot (FIG. 7). The SpA_(KK) variant (harboring lysine substitutions atall 10 glutamine residues) was impaired in its ability to bind Fcγ butnot F(ab)₂ fragments, whereas the SpA_(AA) variant (harboring alaninesubstitutions at all 10 aspartic acid residues) bound to Fcγ but notF(ab)₂ (FIG. 7). The binding of human IgG to SpA was blocked by allthree mAbs (5A10, 3F6 and 3D11) in a manner that exceeded thecompetition of isotype control mAbs (FIG. 4A). All three antibodiesinterfered with the binding of human IgG to SpA_(KK) (Fab binding) or toSpA_(AA) (Fab binding) (FIG. 4A). Thus, SpA_(KKAA)-specific mAbs preventthe non-immune association of SpA with immunoglobulin. Based on thesedata, we presume that protein A-specific mAbs interact withconformational epitopes involving helix 2 of IgBDs, a structural elementinvolved in the Fcγ and Fab interactions of SpA.

If mAb 3F6 binds wild-type SpA as an antigen on the staphylococcalsurface, its Fcγ domain should be available for recognition bycomplement or Fc receptors on the surface of immune cells. To test thisprediction, S. aureus was incubated with 3F6, its isotype control andaffinity purified Sbi₁₋₄. Antibody-mediated co-sedimentation led to thedepletion of soluble Sbi₁₋₄ from the supernatant, which was analyzed asa measure for the availability of FCγ sites on the bacterial surface.Incubation of staphylococci with the control mAb, which can onlyassociate with SpA in a non-immune fashion, caused a modest reduction ofsoluble Sbi₁₋₄ (FIG. 4B). In contrast, incubation of staphylococci withmAb 3F6 depleted soluble Sbi₁₋₄, indicating that mAb 3F6 bound SpAantigen on the bacterial surface while presenting its Fcγ domain forassociation with Sbi₁₋₄ (FIG. 4B).

To test whether the binding of mAb 3F6 to SpA does occur in vivo, BALB/cmice were immunized with mAb 3F6 or the isotype control antibody.Following the injection of purified SpA into the peritoneal cavity, itsabundance in circulation was assessed by sampling blood over the next 30minutes. Compared to animals treated with control mAb, injection of mAb3F6-treated animals caused accelerated clearance of SpA from thebloodstream (FIG. 4C). It is presumed that immune recognition of SpA bymAb 3F6 provides for its Fcγ domain to mediate Fc-receptor mediatedremoval of antigen-antibody complexes from the blood stream.

SpA_(KKAA)-mAbs Promote Opsonopagocytic Killing of Staphylococci inHuman and Mouse Blood.

Eliciting adaptive immune responses that promote opsonophagocytickilling of pathogens is a universal goal for vaccine development andlicensure (Robbins et al., 1996). This has not been achieved for S.aureus, as this pathogen is armed against opsonic antibodies via itssurface exposed and secreted SpA and Sbi molecules (Kim et al., 2011).To test whether SpA_(KKAA)-mAbs can promote opsonophagocytosis, an assayof bacterial killing in fresh blood developed by Rebecca Lancefield wasemployed (Lancefield, 1928). Lepirudin anti-coagulated blood from naïve6 week old BALB/c mice was incubated with MSSA strain Newman in thepresence or absence of 2 μg·ml⁻¹ mAbs 5A10, 3F6 and 3D11 or theirisotype controls. Blood samples were lysed, plated on agar medium andstaphylococcal load enumerated (FIG. 5A). All three mAbs triggeredopsonophagocytic killing of staphylococci, which ranged from 37% of theinoculum (3D11, P=0.0025), to 33% (3F6, P=0.0478) and 16% (5A10,P=0.0280). As a test for opsonophagocytic killing of staphylococci inhuman blood, the inventors recruited healthy human volunteers andexamined their serum for antibodies specific for SpA_(KKAA). As reportedbefore, none of the volunteers harbored serum antibodies directedagainst protein A (data not shown) (Kim et al., 2010a). Anti-coagulatedfresh human blood samples were incubated with MRSA strain USA400 (MW2)in the presence or absence of 10 μg·ml⁻¹ mAbs 5A10, 3F6 and 3D11 ortheir isotype controls (FIG. 5B). All three mAbs triggeredopsonophagocytic killing of staphylococci, which ranged from 52% of theinoculum (3D11, P=0.0002), to 44% (3F6, P=0.0001) and 34% (5A10,P=0.0035). Blood samples were spread on glass slides, stained withGiemsa and analyzed by microscopy. Blood samples incubated in thepresence of mAbs 5A10, 3F6 and 3D11 harbored staphylococci that wereassociated with neutrophils, i.e. they may be associated with theseleukocytes or located within cells (FIG. 5C-E). Blood samples incubatedwith isotype control mAbs harbored clusters of extracellularstaphylococci (red arrowheads) as well as staphylococci that wereassociated with leukocytes (blue arrowheads, (FIG. 5F-H).

Discussion

Monoclonal antibodies offer unique opportunities to investigate thebiological attributes of humoral adaptive immune responses to microbialsurface products, revealing both the molecular nature of microbialimmune evasion and of protective immunity (Fischetti, 1989). Forexample, group A streptococcal M protein, a key virulence factor andα-helical coiled-coil surface protein (Phillips et al., 1981), confersresistance to opsonophagocytic clearance, which may be overcome byhumoral adaptive immune responses during infection (Lancefield, 1962;Scott et al., 1986). mAbs that bind to the α-helical coiled-coil of Mprotein cannot induce opsonophagocytic killing of group A streptococci,which is however achieved by mAbs directed against the N-terminal,random coil domain (Jones and Fischetti, 1988; Jones et al., 1986). TheN-terminal domain of M proteins is highly variable between clinicalisolates, which represents the molecular basis for type-specificimmunity (Hollingshead et al., 1987; Lancefield, 1962).

Similar to streptococcal M protein, protein A also functions as theprotective antigen of S. aureus (Stranger-Jones et al., 2006). Virtuallyall clinical isolates of S. aureus express protein A, however the aminoacid sequence of its IgBDs is highly conserved (McCarthy and Lindsay,2010). Staphylococcal infections in mice or humans do not elicit proteinA-specific humoral immune responses (Kim et al., 2010a), which isexplained by the B cell superantigen activity of this molecule(Silverman and Goodyear, 2006). Immunization with protein A variants, inparticular the SpA_(KKAA) molecule, elicits humoral immune responses inmice and rabbits; these antibodies crossreact with wild-type protein Aand provide protection against staphylococcal disease in mice (Kim etal., 2010a). Affinity purified polyclonal rabbit antibodies can blockthe B cell superantigen activity of the wild-type protein A in mice andenhance the opsonophagocytic capacity of mouse neutrophils whenincubated in anti-coagulated mouse blood. In addition, S. aureus mutantslacking the structural gene for protein A (spa) display significantdefects in virulence, and also permit the development of humoral immuneresponses against many different staphylococcal antigens as well as thedevelopment of protective immunity (Cheng et al., 2009; Kim et al.,2011). Thus, antibodies that neutralize the immune-modulatory attributesof SpA may not only provide protection against acute staphylococcalinfection, but may also enable the development of protective immuneresponses against other staphylococcal antigens and prevent recurrent S.aureus infections. The inventors tested this prediction by raising mAbsagainst SpA_(KKAA). All monoclonal antibodies that elicited protectiveimmunity in mice recognized conformational epitopes of protein A andinteracted with the triple-helical fold of its IgBDs. Importantly, thesemonoclonal antibodies with strong affinity and cross-reactivity formultiple or all IgBDs of SpA_(KKAA) recognized also wild-type protein A.When tested in vitro, mAbs, in particular 5A10, 3F6 and 3D11, preventedprotein A association with the Fcγ and the Fab domains ofimmunoglobulins and triggered opsonophagocytic killing of S. aureus byphagocytes in mouse and human blood. When injected into the peritonealcavity of mice, mAbs elicited significant immune protection against bothMSSA and MRSA S. aureus isolates. Further, SpA_(KKAA)-mAb mediatedneutralization of SpA in vivo stimulated humoral immune responsesagainst several different S. aureus antigens, supporting the hypothesisthat SpA_(KKAA)-antibodies inhibit the B cell superantigen activities ofstaphylococci.

Of note, the magnitude of antibody responses toward staphylococcalantigens in passively immunized mice were much lower than the immuneresponses elicited in mice actively immunized with SpA_(KKAA). The maindifference between active and passive immunization strategies lies inthe development of antigen specific T/B cell populations governing hostimmune responses, which are the consequence of active immunizationstrategies. Future studies are warranted to determine if the protein Aspecific T/B cells are critical in raising appropriate systemic immuneresponses such as T_(H)1/17 mediated recruitment of functionalphagocytes against S. aureus infections (Spellberg et al., 2012).

Previous work demonstrated superantigen activity of protein A towardsV_(H)3-type B cell receptors in mice (Goodyear et al., 2003). Of note,only 5-10% of mouse B cells are V_(H)3-clonal and susceptible to proteinA superantigen (Silverman et al., 2006). Nevertheless, protein A mutantstaphylococci display a profound defect in the pathogenesis of abscessformation in mouse models for this disease (Cheng et al., 2009, Kim etal., 2011). In contrast to mice, human V_(H)3 clonal B cells comprise upto 50% of the total B cell population (Berberian et al., 1993, Huang etal 1992), suggesting that the impact of protein A superantigen activityduring staphylococcal infection is likely greater for human B cellpopulations (Silverman et al., 2006). If so, protein A-mediated B cellactivation may trigger biased use of V_(H)3 B cell clones and thedevelopment of non-physiological B cell populations. Ultimately,staphylococcal protein A is expected to human hosts of V_(H)3 positive Bcells and V_(H)3-type antibodies. A similar scenario is encountered withthe HIV envelope glycoprotein gp120, which also interacts with VH3clonal B cells, causing a clonal deficit of V_(H)3B cells (antibodygenes) in AIDS patients (Berberian et al., 1993, Berberian et al 1991).These events are likely key factors in the prevention of neutralizingantibody responses during HIV and S. aureus infection (Kim et al.,2012b).

Work by others has sought to isolate monoclonal antibodies againstprotein A. One such antibody, SPA27 (Sigma, St. Louis, Mo.) was typed asmouse IgG1, an antibody class whose Fcγ domain does not interact withprotein A (Kronvall et al., 1970). Nevertheless, even IgG1 sub-typeantibodies can be bound by protein A via pseudo-immune associationassuming these antibodies harbor VH3-type Fab domains (Cary et al.,1999, Sasso et al., 1989). We recently developed reagents that candistinguish between these possibilities. For example, wild-type proteinA (SpA) binds immunoglobulins via the Fcγ and the VH3-type Fab domains(Silverman et al., 2006). SpA_(KK) associates only with the Fab domainsof VH3-type antibodies, whereas SpA_(AA) binds only to the Fcγ domain ofIgG but not to the Fab domain of VH3-type immunoglobulin (Kim et al.,2012a). Using these reagents, we observed that SPA27 binds to wild-typeSpA and SpA_(KK), but not to Sp_(AAA) or SpA_(KKAA) (Kim et al., 2012a).Thus, SPA27, which was isolated from a hybridoma following immunizationof mice with wild-type protein A from S. aureus Cowan1, does notspecifically recognize protein A (Kim et al., 2012a). Rather, SPA27 isbound by protein A (Kim et al., 2012a). As could be expected from theseobservations, SPA27 cannot neutralize the IgG or IgM binding activitiesof protein A and it does not provide protection in mice that aresubsequently challenged via S. aureus infection (Kim et al., 2012a).

We wondered whether the inability of the host immune system to produceneutralizing antibodies during S. aureus infection or followingimmunization with wild-type protein A represents a general phenomenon(Kim et al., 2012a, Kim et al 2012b). To test this model, we purifiedmAb 358A76.1, an antibody that was isolated following immunization ofmice with wild-type protein A from S. aureus Cowan1 (Sjoquist et al.,1972) (United States patent US2008/0118937 A1 and US2010/0047252 A1).Unlike mAb SPA27, mAb 358A76.1 displayed immune reactivity with SpA,SpA_(KK), SpA_(AA) and SpA_(KKAA) (Kim et al., 2012a). We observed thatthe specific binding site of mAb 358A76.1 is restricted to E_(KKAA)domain, and that the antibody does not recognize the D_(KKAA), A_(KKAA),B_(KKAA) and C_(KKAA) domains. On the basis of amino acid dissimilaritybetween E_(KKAA) and the other four IgBDs, we presume that mAb 358A76.1recognizes a conformational epitope on the surface of the E-IgBD domain.The association between mAb 358A76.1 and the E domain of protein Acannot neutralize the other four IgBDs (D, A, B and C). Notsurprisingly, passive transfer of mAb 358A76.1 into naïve mice does notconfer protection against S. aureus challenge and does not trigger theopsonophagocytic killing of staphylococci in blood. Based on theseobservations we propose that only the immunization with non-toxigenicprotein A, for example SpA_(KKAA), can elicit the development ofantibodies that neutralize all IgBDs of protein A and that conferprotection against S. aureus disease.

Data presented herein provide corroborating evidence for the generalhypothesis that the neutralization of the Fcγ and Fab binding activitiesof SpA represent a correlate for protective immunity against S. aureus:such antibodies are expected to trigger the opsonophagocytic killing ofthe pathogen in blood and to elicit antibodies that neutralize thesecreted virulence factors of staphylococci (Mazmanian et al., 1999).

Sequence Analysis

Wildtype S. aureus Protein A interacts with human IgG through 2non-antigenic binding sites. The first is with the Fc constant regionand the second is with the Fab heavy chain of the human VH3 clan (theFab binding also occurs with IgA and IgM). Thus, the mAbs that belong tothe mouse clan that corresponds to the human VH3 likely have threepossible, and perhaps competing binding affinities with the wildtypeantigen, one for Fc, one for Fab, and the third the antigen specificbinding mediated via the CDRs. Hybridoma cell lines that generateSpAKKAA-specific monoclonal antibodies were subjected to CDR sequencinganalysis. Each individual antibody included two antigen recognitionssites (Fab fragments) wherein each Fab portion comprised three CDRs inthe light chain and three CDRs in the heavy chain. The mAbs identifiedas conferring protection against S. aureus infection include 5A10, 3F6,3D11, 5A11, 1B10 and 4C1.

3F6, which provided significant protection, presented the mostdistinctive sequence among a panel of SpAkkaa mAbs. Despite sharing thesame CDR sequence of the light chain with 1F10 and 6D11, 3F6 has aunique CDR sequence in the heavy chain that distinguishes it from 1F10and 6D11. Finally, 1F10 and 6D11 shared common heavy and light chain CDRsequences, suggesting they are sibling mAbs. Both the 1F10 and 6D11antibodies failed to generate significant immune protection in mice. Thethree mAbs that produced the most promising protective effects and thathave been further characterized (5A10, 3F6, and 2F2) are indicated inbold

Three main groups of light chain sequences were identified, united bysimilar sequences. A first group comprised 3F6, 1F10, 6D11, 4C1, 6B2,2B8, and 4C5, all of which shared common light chain CDR sequences, withthe exception of one amino acid difference in 6B2 and two amino aciddifferences in 4C5. Despite sharing light chain sequences, these mAbsproduced a variety of protective effects, suggesting that specificdifferences in the heavy chain sequences may significantly influence thefunctional effects of these mAbs. A second group shared a set of lightchain CDR sequences (the heavy chain CDR sequences differed) andcomprised 5A10 and 2F2, including one of the main protective antibodies,5A10.

The percent identity of the corresponding CDRs of all antibodies werecalculated and are presented in Tables 7-15 in a matrix format. Theantibodies that had greater than 40% individual CDR sequence identitywith respect to the individual CDRs of 3F6, 5A10, or 3D11 are summarizedin Table 16. Consensus sequences for each set of CDRs that were greaterthan 40% identical to the corresponding CDRs of 3F6 are presented inTable 17.

TABLE 5  Antibody sequences. ^(e)CFU b ^(c)Heavy Chain Sequence^(d)Light Chain Sequence reduction/ CDR1 CDR2 CDR3 CDR1 CDR2 CDR3^(f)Abcess ^(b)V_(H) Amino acid sequence Amino acid sequence Formation Pclan Nucleotide sequence Nucleotide sequence values IgG1 5A10 V_(H)3GFAFSNYD ISSGGTYP ARGGFLITT SSVSY DTS QQWSSYPPT 0.0019/ (SEQ ID (SEQ IDRDYYAMDY (SEQ ID (SEQ ID (SEQ ID 0.035 NO: 11) NO: 12) (SEQ ID NO: 16)NO: 17) NO: 18) NO: 13) EVKLVESGGGLVKPGGSLKLSCAASG FAFSNYDTIVLTQSPAIMSASPGEKVTMTCSAS SSVS YMYWYQQKPG MSWVRQTPEKRLEWVAT ISSGGTYPYYPDSVKG SSPRLLIY DTS NLASGVPVRFSGSGSGTSYSLTISRMEAERFTISRDNAKNTLYLQLSSLRSEDTALYYC ARG DAATYYC QQWSSYPPT FGGGTKLEIKGFLITTRDYYAMDY WGQGTSVTVSS (SEQ ID NO: 14) (SEQ ID NO: 19)GaagtgaagctggtggagtctgggggaggcttaAcaattgttctcacccagtctccagcaatcatgtctgcatgtgaagcctggagggtccctgaaactctcctgtctccaggggagaaggtcaccatgacctgcagtgccagctcgcagcctccggattcgctttcagtaactatgacaagtgtaagttacatgtactggtaccagcagaagccaggaatgtcttgggttcgccagactccggagaagagg ctggagtgggtcgcaaccattagtagtggtggttcctcccccagactcctgatttatgacacatccaacctggacttacccctactatccagacagtgtgaagggccttctggagtccctgttcgcttcagtggcagtgggtctggcgtttcaccatctccagagacaatgccaagaacgacctcttactctctcacaatcagccgaatggaggctgaaaccctgtacctgcaattgagcagtctgaggtct gaggacacggccttgtattactgtgcaagaggggatgctgccacttattactgccagcagtggagtagttaccggatttttgattacgacacgggattactatgctcacccacgttcggaggggggaccaagctggaaataaaacatggactactggggtcaaggaacctcagtcacc (SEQ ID NO: 20)gtctcctcag (SEQ ID NO: 15) 8E2 V_(H)1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 11) NO: 12) (SEQ ID NO: 16) NO: 17) NO: 18) NO: 13)

Example 2 Materials and Methods

Bacterial Strains and Growth Conditions.

S. aureus strains Newman and MW2 were grown in tryptic soy broth (TSB)at 37° C. Escherichia coli strains DH5a and BL21 (DE3) were grown inLuria-Bertani (LB) broth with 100 μg·ml-1 ampicillin at 37° C.

Monoclonal Antibodies.

Mouse monoclonal antibodies were generated by the conventional method(Köhler, G., and C. Milstein. 1975). Briefly, BALB/c mice (8 week old,female, Jackson Laboratory) were immunized by intraperitoneal injectionwith 100 μg purified SpA_(KKAA) emulsified 1:1 with Complete Freund'sAdjuvant (CFA, DIFCO). On days 21 and 42, mice were boosted byintraperitoneal injection with 100 μg of the same antigen emulsified 1:1with Incomplete Freund's Adjuvant (IFA, DIFCO). On days 31 and 52, micewere bled and serum samples screened by ELISA for specific antibodies.Seventy-nine days following initial immunization, mice that demonstratedstrong antigen-immunoreactivity by ELISA were boosted with 25 μg of thesame antigen. Three days later, splenocytes were harvested and fusedwith the mouse myeloma cell line SP2/mL-6, an interleukin 6 secretingderivative of the SP2/0 myeloma cell line. Supernatants from resultinghybridomas were screened by ELISA and antigen-specific clones werefurther subcloned by limiting dilution to yield monoclonalantibody-secreting hybridomas arising from single cells. Antibodies werepurified from the spent culture supernatant of cell lines. Spa27monoclonal antibody was purchased from Sigma. Hybridoma cell line358A76.1.1 was purchased from American Type Culture Collection (ATCCaccession number PTA-7938) and expanded at the Fitch monoclonal antibodyfacility (University of Chicago).

Purification of Recombinant Proteins.

Polypeptides derived from the amino acid sequence of the SpA-E_(KKAA)domain were synthesized by CPC Scientific Inc (Sunnyvale, USA).Lyophilized peptide samples were solubilized using either distilledwater or dimethyl sulfoxide (DMSO), then aliquoted and frozen at −80° C.The use of plasmids for wild-type SpA and SpA_(KKAA) has been previouslydescribed (Kim et al., 2010a). Oligonucleotides for the synthesis ofSpA_(KK) (Q⁹K, Q¹⁰K substitutions in each of the five IgBDs), SpA_(AA)(D³⁶A, D³⁷A substitutions in each of the five IgBDs), individual IgBDs(E, D, A, B and C) of SpA_(KKAA) were synthesized by Integrated DNATechnologies, Inc (USA). PCR products of SpA_(KKAA) variants were clonedinto the pET15b vector generating N-terminal His₆-tagged recombinantproteins. The coding sequence of Sbi₁₋₄ was PCR amplified with twoprimers, 5′-AAAAAAGCTAGCTGGTCTCATCCTCAATTTGAGAAGACGCAACAAACTTCAACTAAG-3′ (SEQ ID NO:8) and 5′-AAAAAACTCGAGTTTCCAGAATGATAATAAATTAC-3′ (SEQ IDNO:9) from S. aureus Newman chromosomal DNA with engineered N-terminalStrep tag (WSHPQFEK (SEQ ID NO:10)). PCR product of Sbi₁₋₄ was clonedinto pET24b vector generating C-terminal His₆-tagged recombinant proteinwith engineered N-terminal Strep tag (WSHPQFEK (SEQ ID NO:10)). Allplasmids were transformed into BL21(DE3) for affinity purification.Overnight cultures of recombinant E. coli strains were diluted 1:100into fresh media and grown at 37° C. to A₆₀₀ 0.5, at which pointcultures were induced with 1 mM isopropyl β-D-1-thiogalatopyranoside(IPTG) and grown for an additional three hours. Bacterial cells weresedimented by centrifugation, suspended in column buffer (50 mM Tris-HCl(pH 7.5), 150 mM NaCl) and disrupted with a French pressure cell at14,000 psi. Lysates were cleared of membrane and insoluble components byultracentrifugation at 40,000×g. Proteins in the cleared lysate weresubjected to nickel-nitrilotriacetic acid (Ni-NTA) affinitychromatography. Proteins were eluted in column buffer containingsuccessively higher concentrations of imidazole (100-500 mM). Proteinconcentrations were determined by bicinchonic acid (BCA) assay (ThermoScientific).

Enzyme Linked Immunosorbent Assay.

To determine SpA specific serum IgG, affinity purified SpA_(KKAA) wasused to coat ELISA plates (NUNC Maxisorp) at 1 μg·ml⁻¹ in 0.1 Mcarbonate buffer (pH 9.5 at 4° C.) overnight. The following day, plateswere blocked and incubated with dilutions of hyperimmune sera anddeveloped using OptEIA reagent (BD Biosciences). For the determinationof binding affinity of SpA-specific mAbs, ELISA plates were coated withaffinity purified individual immunoglobulin binding domains or syntheticpeptides (H1, H2, H3, H1+3 and H2+3) whose sequences were derived fromthe sequence of SpA-E_(KKAA) Peptides were used for plate coating at aconcentration of 100 nM in 0.1 M carbonate buffer, pH 9.5 at 4° C.overnight. The following day, plates were blocked with 1% BSA solutionin PBS-T and incubated with variable concentrations of SpA-specificmAbs. To determine the avidity of specific mAbs, antibody-antigeninteractions were perturbed with increasing concentration (0-4 M) ofammonium thiocyanate. For SpA and Sbi binding assays, affinity purifiedSpA and Sbi were coated onto ELISA plate at 1 μg·ml⁻¹ in 0.1 M carbonatebuffer (pH 9.5 at 4° C.) overnight. The following day, plates wereblocked and incubated with dilutions of peroxidase-conjugated human IgG,Fc and F(ab)₂ (The Jackson Laboratory) or dilutions of isotype controlantibodies and SpA_(KKAA)-specific mAbs; assays were developed usingOptEIA reagent. To measure the inhibition of immune association betweenhuman IgG and SpA, plates were incubated with either 20 μg·ml-1 isotypecontrol antibodies or SpA_(KKAA)-specific mAbs prior to ligand binding.For competition assay, plates were coated with 10 ng·ml⁻¹ SpA_(KKAA) in0.1 M carbonate buffer (pH 9.5) at 4° C. overnight. The following day,plates were blocked and incubated with 30 μg·ml⁻¹ of isotype controlantibodies or SpA_(KKAA)-specific mAbs prior to the incubation withHRP-conjugated SpA-specific mAbs (Innova Biosciences) or human IgG at afinal concentration of 100-200 ng·ml⁻¹.

Mouse Renal Abscess Model.

Affinity purified antibodies in PBS were injected at a concentration 5,15, 20, or 50 mg·kg⁻¹ of experimental animal weight into the peritonealcavity of BALB/c mice (6 week old, female, Charles River Laboratories)4-24 hours prior to challenge with S. aureus. Overnight cultures of S.aureus strains were diluted 1:100 into fresh TSB and grown for 2 hoursat 37° C. Staphylococci were sedimented, washed and suspended in PBS tothe desired bacterial concentration. Inocula were quantified byspreading sample aliquots on TSA and enumerating the colonies thatformed upon incubation. BALB/c mice were anesthetized viaintraperitoneal injection with 100 mg·ml⁻¹ ketamine and 20 mg·ml⁻¹xylazine per kilogram of body weight. Mice were infected by injectionwith 1×10⁷ CFU of S. aureus Newman or 5×10⁶ CFU of S. aureus USA300(LAC) or USA400 (MW2) into the periorbital venous sinus of the righteye. On day 4 or 15 following challenge, mice were killed by CO₂inhalation. Both kidneys were removed, and the staphylococcal load inone organ was analyzed by homogenizing renal tissue with PBS, 0.1%Triton X-100. Serial dilutions of homogenate were spread on TSA andincubated for colony formation. The remaining organ was examined byhistopathology. Briefly, kidneys were fixed in 10% formalin for 24 hoursat room temperature. Tissues were embedded in paraffin, thin-sectioned,stained with hematoxylin-eosin, and inspected by light microscopy toenumerate abscess lesions. Immune serum samples collected at 15 dayspost infection were examined by immunoblotting against 14 affinitypurified staphylococcal antigens immobilized onto nitrocellulosemembrane at 2 μg. Signal intensities were quantified as previouslydescribed (Kim et al., 2010b). All mouse experiments were performed inaccordance with the institutional guidelines following experimentalprotocol review and approval by the Institutional Biosafety Committee(IBC) and the Institutional Animal Care and Use Committee (IACUC) at theUniversity of Chicago.

Staphylococcal Survival in Blood.

Whole blood was collected from BALB/c mice by cardiac puncture andcoagulation inhibited with 10 μg·ml⁻¹ lepirudin. 50 μl of 5×10⁵ CFU·ml⁻¹of S. aureus Newman were mixed with 950 μl of mouse blood in thepresence of 2 μg·ml⁻¹ of mAbs. Samples were incubated at 37° C. withslow rotation for 30 minutes and then incubated on ice with 1%saponin/PBS. For human blood studies, 50 μl of 5×10⁶ CFU ml⁻¹ of S.aureus MW2 were mixed with 950 μl of freshly drawn human blood in thepresence of 10 μg·ml⁻¹ of mAbs. The tubes were incubated at 37° C. withslow rotation for 120 minutes. Aliquots were incubated on ice with 1%saponin/PBS to lyse blood cells. Dilutions of staphylococci were platedon agar for colony formation. Experiments with blood from humanvolunteers were performed with protocols that had been reviewed,approved, and supervised by the University of Chicago's InstitutionalReview Board (IRB).

SpA-Specific Serum IgG.

BALB/c mice were injected into the peritoneum with 20 μg affinitypurified SpA variants in the presence of 85 μg mAb 3F6 or its isotypecontrol at day 0 and 11. At day 21, whole blood was collected fromBALB/c mice to obtain hyperimmune sera.

Measuring the Abundance SpA in Circulation.

Passively immunized BALB/c mice were injected into the peritoneum with200 μg affinity purified wild-type SpA. At indicated time intervals,whole blood was collected from BALB/c mice with 10 μg·ml⁻¹ of lepirudinanticoagulant. All samples were kept on ice with 1% saponin/PBS for 10minutes. Lysed samples were then diluted in 1:10 PBS and mixed withSDS-PAGE sample buffer in 1:1. Samples were boiled for 5 minutes at 90°C. prior to SDS-PAGE gel electrophoresis. Samples were transferred toPDVF and analyzed by immunoblotting with affinity-purified rabbitα-SpA_(KKAA) antibody.

Sbi Consumption Assay.

Overnight cultures of S. aureus Newman were diluted 1:100 into freshTSB, grown for 2 hours and A600 adjusted to 0.4 (1×10⁸ CFU·ml⁻¹) withpre-chilled TSB. Cells were washed and incubated with either 100 μl ofisotype control or mAb 3F6 at a final concentration of 100 μg·ml⁻¹ foran hour at 4° C. Following incubation, staphylococci were washed withpre-chilled TSB and incubated with 2 μg of affinity-purified wild-typeSbi for one hour at 4° C. Staphylococci were sedimented bycentrifugation at 13,000×g for one minute, supernatants were removed andmixed with sample buffer (1:1). Samples were boiled for 5 minutes at 90°C. prior to SDS-PAGE gel electrophoresis. Samples wereelectrotransferred to PDVF membrane and analyzed by immunoblotting withaffinity-purified rabbit α-SpA_(KKAA) antibody.

Sequencing of Monoclonal Antibodies.

Total RNA samples from hybridoma cells were isolated using astandardized protocol. Briefly, 1.4×10⁷ hybridoma cells cultured inDMEM-10 medium with 10% FBS were washed with PBS, sedimented bycentrifugation and lysed in TRIzol (Invitrogen). Samples were mixed with20% chloroform and incubated at room temperature for three minutes andcentrifuged at 10,000×g for fifteen minutes at 4° C. RNAs in the aqueouslayer were removed and washed with 70% isopropanol. RNA was sedimentedby centrifugation and washed with 75% diethylpyrocarbonate(DEPC)-ethanol. Pellets were dried and RNA dissolved in DEPC. cDNA wassynthesized with the cDNA synthesis kit (Novagen) and PCR amplifiedusing the PCR Reagent System (Stratagene), independent primers (5 pmoleach) and a mouse variable heavy and light chain specific primer set(Novagen). PCR products were sequenced and analyzed using IMGT Vquest(available at imgt.cines.fr/IMGT_vquest).

Statistical Analysis.

Bacterial loads and number of abscesses in the experimental animal modelfor S. aureus infection were analyzed with the two-tailed Mann-Whitneytest to measure statistical significance. Unpaired two-tailed Student'st-tests were performed to analyze the statistical significance of ELISAdata, immunoblotting signals, and ex vivo blood survival data. All datawere analyzed by Prism (GraphPad Software, Inc.) and P values less than0.05 were deemed significant.

TABLE 7 (Variable Light chain CDR percent identity matrix)  1: 5A10 10029 29 29 41 41 41 69 100 41 41 41 35  2: 8E2 29 100 17 56 28 28 28 25 2928 28 28 28  3: 3A6 29 17 100 22 32 32 32 31 29 32 32 32 32  4: 7E2 2956 22 100 28 28 28 19 29 28 28 28 28  5: 3F6 41 28 32 28 100 100 100 3841 100 95 100 91  6: 1F10 41 28 32 28 100 100 100 38 41 100 95 100 91 7: 6D11 41 28 32 28 100 100 100 38 41 100 95 100 91  8: 3D11 69 25 3119 38 38 38 100 69 38 38 38 31  9: 2F2 100 29 29 29 41 41 41 69 100 4141 41 35 10: 4C1 41 28 32 28 100 100 100 38 41 100 95 100 91 11: 6B2 4128 32 28 95 95 95 38 41 95 100 95 86 12: 2B8 41 28 32 28 100 100 100 3841 100 95 100 91 13: 4C5 35 28 32 28 91 91 91 31 35 91 86 91 100

TABLE 8 (Variable Heavy chain CDR percent identity matrix) 5A10 1B10 2C38E2 3A6 7E2 3F6 1F10 6D11 3D11 5A11 2F2 8D4  1: 5A10 100 97 97 42 24 2738 32 32 40 45 38 26  2: 1B10 97 100 100 45 24 31 41 28 28 40 48 41 30 3: 2C3 97 100 100 45 24 31 41 28 28 40 48 41 30  4: 8E2 42 45 45 100 3338 33 40 40 53 28 25 30  5: 3A6 24 24 24 33 100 62 19 30 30 33 29 14 24 6: 7E2 27 31 31 38 62 100 24 32 32 32 31 24 26  7: 3F6 38 41 41 33 1924 100 17 17 28 30 24 27  8: 1F10 32 28 28 40 30 32 17 100 100 36 32 2533  9: 6D11 32 28 28 40 30 32 17 100 100 36 32 25 33 10: 3D11 40 40 4053 33 32 28 36 36 100 32 22 35 11: 5A11 45 48 48 28 29 31 30 32 32 32100 41 30 12: 2F2 38 41 41 25 14 24 24 25 25 22 41 100 9 13: 8D4 26 3030 30 24 26 27 33 33 35 30 9 100

TABLE 9 (Variable Light and Heavy chain CDR percent identity) 5A10 8E23A6 7E2 3F6 1F10 6D11 3D11 2F2 1: 5A10 100 38 26 28 39 33 33 54 61 2:8E2 38 100 26 45 31 33 33 46 27 3: 3A6 26 26 100 44 26 30 30 32 21 4:7E2 28 45 44 100 25 29 29 29 26 5: 3F6 39 31 26 25 100 57 57 33 30 6:1F10 33 33 30 29 57 100 100 38 34 7: 6D11 33 33 30 29 57 100 100 38 348: 3D11 54 46 32 29 33 38 38 100 44 9: 2F2 61 27 21 26 30 34 34 44 100

TABLE 10 (Variable Light Chain CDR1 percent identity matrix) 5A10 8E23A6 7E2 3F6 1F10 6D11 3D11 2F2 4C1 6B2 2B8 4C5  1: 5A10 100 0 40 0 60 6060 100 100 60 60 60 60  2: 8E2 0 100 17 50 17 17 17 0 0 17 17 17 33  3:3A6 40 17 100 17 20 20 20 40 40 20 20 20 20  4: 7E2 0 50 17 100 17 17 170 0 17 17 17 33  5: 3F6 60 17 20 17 100 100 100 60 60 100 90 100 90  6:1F10 60 17 20 17 100 100 100 60 60 100 90 100 90  7: 6D11 60 17 20 17100 100 100 60 60 100 90 100 90  8: 3D11 100 0 40 0 60 60 60 100 100 6060 60 60  9: 2F2 100 0 40 0 60 60 60 100 100 60 60 60 60 10: 4C1 60 1720 17 100 100 100 60 60 100 90 100 90 11: 6B2 60 17 20 17 90 90 90 60 6090 100 90 80 12: 2B8 60 17 20 17 100 100 100 60 60 100 90 100 90 13: 4C560 33 20 33 90 90 90 60 60 90 80 90 100

TABLE 11 (Variable Light Chain CDR2 percent identity matrix) 5A10 8E23A6 7E2 3F6 1F10 6D11 3D11 2F2 4C1 6B2 2B8 4C5  1: 5A10 100 0 33 0 33 3333 33 100 33 33 33 33  2: 8E2 0 100 0 67 33 33 33 0 0 33 33 33 33  3:3A6 33 0 100 0 33 33 33 33 33 33 33 33 33  4: 7E2 0 67 0 100 33 33 33 00 33 33 33 33  5: 3F6 33 33 33 33 100 100 100 33 33 100 100 100 100  6:1F10 33 33 33 33 100 100 100 33 33 100 100 100 100  7: 6D11 33 33 33 33100 100 100 33 33 100 100 100 100  8: 3D11 33 0 33 0 33 33 33 100 33 3333 33 33  9: 2F2 100 0 33 0 33 33 33 33 100 33 33 33 33 10: 4C1 33 33 3333 100 100 100 33 33 100 100 100 100 11: 6B2 33 33 33 33 100 100 100 3333 100 100 100 100 12: 2B8 33 33 33 33 100 100 100 33 33 100 100 100 10013: 4C5 33 33 33 33 100 100 100 33 33 100 100 100 100

TABLE 12 (Variable Light Chain CDR3 percent identity matrix) 5A10 8E23A6 7E2 3F6 1F10 6D11 3D11 2F2 4C1 6B2 2B8 4C5  1: 5A10 100 25 33 50 4444 44 88 100 44 44 44 44  2: 8E2 25 100 25 56 25 25 25 25 25 25 25 25 25 3: 3A6 33 25 100 25 44 44 44 38 33 44 44 94 44  4: 7E2 50 56 25 100 2525 25 50 50 25 25 25 25  5: 3F6 44 25 44 25 100 100 100 50 44 100 100100 89  6: 1F10 44 25 44 25 100 100 100 50 44 100 100 100 89  7: 6D11 4425 44 25 100 100 100 50 44 100 100 100 89  8: 3D11 88 25 38 50 50 50 50100 88 50 50 50 50  9: 2F2 100 25 33 50 44 44 44 88 100 44 44 44 44 10:4C1 44 25 44 25 100 100 100 50 44 100 100 100 89 11: 6B2 44 25 44 25 100100 100 50 44 100 100 100 89 12: 2B8 44 25 44 25 100 100 100 50 44 100100 100 89 13: 4C5 44 25 44 25 89 89 89 50 44 89 89 89 100

TABLE 13 (Variable Heavy Chain CDR1 percent identity matrix) 5A10 8E23A6 7E2 3F6 1F10 6D11 3D11 2F2 1B10 2C3 5A11 8D4  1: 5A10 100 38 38 3838 62 62 38 50 88 88 62 38  2: 8E2 38 100 75 88 38 50 50 62 38 50 50 5050  3: 3A6 38 75 100 88 25 50 50 62 25 38 38 50 38  4: 7E2 38 88 88 10038 50 50 62 38 50 50 62 50  5: 3F6 38 38 25 38 100 25 25 25 38 50 50 5038  6: 1F10 62 50 50 50 25 100 100 50 25 50 50 38 50  7: 6D11 62 50 5050 25 100 100 50 25 50 50 38 50  8: 3D11 38 62 62 62 25 50 50 100 38 3838 50 38  9: 2F2 50 38 25 38 38 25 25 38 100 62 62 62 25 10: 1B10 88 5038 50 50 50 50 38 62 100 100 75 50 11: 2C3 88 50 38 50 50 50 50 38 62100 100 75 50 12: 5A11 62 50 50 62 50 38 38 50 62 75 75 100 38 13: 8D438 50 38 50 38 50 50 38 25 50 50 38 100

TABLE 14 (Variable Heavy Chain CDR2 percent identity matrix) 5A10 8E23A6 7E2 3F6 1F10 6D11 3D11 2F2 1B10 2C3 5A11 8D4  1: 5A10 100 25 12 2525 25 25 20 62 100 100 75 20  2: 8E2 25 100 0 12 22 50 50 40 12 25 25 2520  3: 3A6 12 0 100 62 12 25 25 40 12 12 12 12 20  4: 7E2 25 12 62 10012 38 38 40 12 25 25 12 20  5: 3F6 25 22 12 12 100 25 25 0 38 25 25 12 0 6: 1F10 25 50 25 38 25 100 100 40 25 25 25 38 40  7: 6D11 25 50 25 3825 100 100 40 25 25 25 38 40  8: 3D11 20 40 40 40 0 40 40 100 20 20 2020 33  9: 2F2 62 12 12 12 38 25 25 20 100 62 62 50 0 10: 1B10 100 25 1225 25 25 25 20 62 100 100 75 20 11: 2C3 100 25 12 25 25 25 25 20 62 100100 75 20 12: 5A11 75 25 12 12 12 38 38 20 50 75 75 100 60 13: 8D4 20 2020 20 0 40 40 33 0 20 20 60 100

TABLE 15 (Variable Heavy Chain CDR3 percent identity matrix) 5A10 8E23A6 7E2 3F6 1F10 6D11 3D11 2F2 1B10 2C3 5A11 8D4  1: 5A10 100 42 0 12 430 0 36 15 100 100 12 0  2: 8E2 42 100 0 10 44 33 33 67 25 42 42 30 0  3:3A6 0 0 100 0 40 0 0 20 20 0 0 0 50  4: 7E2 12 10 0 100 20 33 33 20 0 1212 20 0  5: 3F6 43 44 40 20 100 20 20 43 9 43 43 20 29  6: 1F10 0 33 033 20 100 100 20 0 0 0 22 0  7: 6D11 0 33 0 33 20 100 100 20 0 0 0 22 0 8: 3D11 36 67 20 20 43 20 20 100 27 36 36 20 29  9: 2F2 15 25 20 0 9 00 27 100 15 15 14 14 10: 1B10 100 42 0 12 43 0 0 36 15 100 100 12 0 11:2C3 100 42 0 12 43 0 0 36 15 100 100 12 0 12: 5A11 12 30 0 20 20 22 2220 14 12 12 100 0 13: 8D4 0 0 50 0 29 0 0 29 14 0 0 0 100

TABLE 16 List of antibodies in which the indicated CDR has 40% orgreater sequence identity to the indicated reference antibody. 5A10 40%or greater identity with reference to 5A10: L CDR1 3A6, 3F6, 1F10, 6D11,3D11, 2F2, 4C1, 6B2, 2B8, 4C5 L CDR2 2F2 L CDR3 7E2, 3F6, 1F10, 6D11,3D11, 2F2, 4C1, 6B2, 2B8, 4C5 H CDR1 1F10, 6D11, 2F2, 1B10, 2C3, 5A11 HCDR2 2F2, 1B10, 2C3, 5A11 H CDR3 8E2, 3F6, 1B10, 2C3 3F6 40% or greateridentity with reference to 3F6: L CDR1 5A10, 1F10, 6D11, 3D11, 2F2, 4C1,6B2, 2B8, 4C5 L CDR2 1F10, 6D11, 4C1, 6B2, 2B8, 4C5 L CDR3 5A10, 3A6,1F10, 6D11, 3D11, 2F2, 4C1, 6B2, 2B8, 4C5 H CDR1 1B10, 2C3, 5A11 H CDR2— H CDR3 5A10, 8E2, 3A6, 3D11, 1B10, 2C3 3D11 40% or greater identitywith reference to 3D11: L CDR1 5A10, 3A6, 3F6, 1F10, 6D11, 2F2, 4C1,6B2, 2B8, 4C5 L CDR2 — L CDR3 5A10, 7E2, 3F6, 1F10, 6D11, 2F2, 4C1, 6B2,2B8, 4C5 H CDR1 8E2, 3A6, 7E2, 1F10, 6D11, 5A11 H CDR2 8E2, 3A6, 7E2,1F10, 6D11 H CDR3 8E2, 3F6

TABLE 17  (Consensus sequences for the CDRs of antibodies with 40% orgreater identity with respect to 3F6) Light Chain CDR1 100%ESVEYSGASL (SEQ ID NO: 145)  90% ESV[E,D]YSGASL (SEQ ID NO: 146)  60%[E,-][S,-][V,-][E,-][Y,-]S[G,S][A,V]S[L,Y] (SEQ ID NO: 147) Overall[E,S]SV[E,D,S]Y[S,Y]GASL (SEQ ID NO: 148) consensus Light Chain CDR2100% AAS (SEQ ID NO: 149) Light Chain CDR3 100%QQSRKVPST (SEQ ID NO: 150)  89% QQSRKVP[S,N]T (SEQ ID NO: 151)  50%QQ[S,W][R,S][K,Y][V,P][P,-][S,F]T (SEQ ID NO: 152)  44%[Q,S]Q[S,W,I][R,S,T][K,Y,S][V,Y]P[SS,P,W]T (SEQ ID NO: 153) Overall[Q,S]Q[S,W,I][R,S,T][K,S,Y][V,Y]P[S,P,W,F,N] consensus (SEQ ID NO: 154)Heavy Chain CDR1 100% GFTFNTNA (SEQ ID NO: 155)  50%GFTF[N,S][T,N,D][N,Y][A,D,Y] (SEQ ID NO: 156) OverallGFTF[S,N][T,N,D][N,Y][A,D,Y] (SEQ ID NO: 157) consensus Heavy Chain CDR2100% IRSKSNNYAT (SEQ ID NO: 158) Heavy Chain CDR3 OverallRH[A,G][R,Y]G[V,G,N,A][T,F,R][E,L,A,Y][H,I,G] consensus[Y,T,F][D,T][Y,R,C,G][D,V,T][Y,G,F]Y[V,A]MDY (SEQ ID NO: 159)

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method of treating or preventing aStaphylococcus infection comprising administering to a patient having aStaphylococcus infection or at risk of a Staphylococcus infection aneffective amount of a purified SpA binding polypeptide that is capableof specifically binding at least two SpA IgG binding domains A, B, C, Dand E of a Spa variant that lacks non-specific Ig-binding activity. 2.The method of claim 1, wherein the purified SpA binding polypeptidebinds to at least two and up to five Spa IgG binding domains A_(KKAA),B_(KKAA), C_(KKAA), D_(KKAA) and E_(KKAA).
 3. The method of claim 2,wherein the purified Spa binding polypeptide has an association constantof 0.5×10⁹ M⁻¹ or greater for at least two and up to five Spa IgGbinding domains A_(KKAA), B_(KKAA), C_(KKAA), D_(KKAA) and E_(KKAA). 4.The method of any of claims 1-3, wherein the purified SpA bindingpolypeptide is capable of reducing staphylococcal load in the patient.5. The method of any of claims 1-4, wherein the antibody is capable ofmediating opsonophagocytic killing of S. aureus.
 6. The method of any ofclaims 1-5, wherein the antibody is capable of perturbing the binding ofhuman IgG to wild-type Spa.
 7. The method of any of claims 1-6, whereinthe purified SpA binding polypeptide is a humanized antibody comprisingat least one amino acid region that is at least 40% identical to a CDRamino sequence from the 5A10, 8E2, 3A6, 3F6, 1F10, 6D11, 3D11, 5A11,1B10, 4C1, 2F2, 4C5, 4D5, 6B2, 8D4, 2B8, 2C3 or 7E2 monoclonalantibodies.
 8. The method of any of claims 1-7, wherein treating aStaphylococcus infection comprises reducing abscess formation orreducing bacterial load in the patient.
 9. The method of claims 1-8,wherein the SpA polypeptide that lacks non-specific Ig-bind bindingactivity is SpA_(KKAA).
 10. The method of any of claims 1-9, wherein thepurified Spa binding polypeptide competes for binding of SpA_(KKAA)polypeptide with the 5A10, 8E2, 3A6, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10,4C1, 2F2, 4C5, 4D5, 6B2, 8D4, 2B8, 2C3 or 7E2 monoclonal antibodies. 11.The method of any of claims 1-10, wherein the antibody has anassociation constant for the SpA_(KKAA) polypeptide of between about 0.5and 100×10⁹ M⁻¹, 1.0 and 100×10⁹ M⁻¹, or 2.0 and 100×10⁹ M⁻¹ as measuredby ELISA.
 12. The method of any of claims 1-11, further comprisingadministering an effective amount of two or more purified Spa bindingpolypeptides.
 13. The method of any of claims 1-12, wherein the purifiedSpa binding polypeptide is recombinant.
 14. The method of claim 13,wherein the recombinant polypeptide is a single domain antibody.
 15. Themethod of any of claim 14, wherein the purified Spa binding polypeptideis a humanized antibody.
 16. The method of any of claims 1-15, whereinthe purified Spa binding polypeptide is a human antibody.
 17. The methodof any of claims 1-16, wherein the purified polypeptide is a recombinantpolypeptide comprising one or more CDR domain from a SpA-bindingantibody.
 18. The method of claim 17, wherein the recombinantpolypeptide comprises one or more CDR domain from the 5A10, 8E2, 3A6,3F6, 1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 4C5, 4D5, 6B2, 8D4, 2B8,2C3 or 7E2 monoclonal antibodies.
 19. The method of claim 18, whereinthe recombinant polypeptide comprises two or more CDR domains from the5A10, 8E2, 3A6, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 4C5, 4D5,6B2, 8D4, 2B8, 2C3 or 7E2 monoclonal antibodies
 20. The method of claim18, wherein the recombinant polypeptide comprises three CDR domains fromamong the VH or VL domain of the 5A10, 8E2, 3A6, 3F6, 1F10, 6D11, 3D11,5A11, 1B10, 4C1, 2F2, 4C5, 4D5, 6B2, 8D4, 2B8, 2C3 or 7E2 monoclonalantibodies.
 21. The method of any of claims 1-20, wherein the purifiedpolypeptide comprises a sequence at least 40% identical to the VH or VLdomain of the 5A10, 8E2, 3A6, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10, 4C1,2F2, 4C5, 4D5, 6B2, 8D4, 2B8, 2C3 or 7E2 monoclonal antibodies.
 22. Themethod of claim 20, wherein the recombinant polypeptide comprises sixCDR domains from among the VH and VL domains of the 5A10, 8E2, 3A6, 3F6,1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 4C5, 4D5, 6B2, 8D4, 2B8, 2C3 or7E2 monoclonal antibodies.
 23. The method of any of claims 1-22, whereinthe purified polypeptide comprises the VH domain from the 5A10, 8E2,3A6, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 4C5, 4D5, 6B2, 8D4,2B8, 2C3 or 7E2 monoclonal antibodies.
 24. The method of any of claims1-23, wherein the purified polypeptide comprises the VL domain the 5A10,8E2, 3A6, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 4C5, 4D5, 6B2,8D4, 2B8, 2C3 or 7E2 monoclonal antibodies.
 25. The method of claim 17,wherein the recombinant polypeptide comprises one or more CDR domainfrom a SpA-binding antibody and a scaffold from a polypeptide selectedfrom the group consisting of an immunoglobulin, a fibronectin, alipocalin or a S. aureus protein Z.
 26. The method of claim 13, whereinthe recombinant antibody segment is operatively coupled to a secondrecombinant antibody segment.
 27. The method of claim 26, wherein thesecond recombinant antibody segment binds a second Staphylococcalprotein.
 28. The method of claim 1, wherein the antibody is a 5A10, 8E2,3A6, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 4C5, 4D5, 6B2, 8D4,2B8, 2C3 or 7E2 monoclonal antibodies.
 29. The method of any of claims1-28, further comprising administering a second antibody that binds asecond Staphylococcal protein.
 30. The method of any of claims 1-29,further comprising administering an antibiotic or a Staphylococcalvaccine composition.
 31. The method of any of claims 1-30, wherein theantibody is administered at a dose of 0.1 mg/kg to 500 mg/kg.
 32. Apurified polypeptide comprising an amino acid sequence that is at least40% identical to one or more antibody CDR domains from a SpA-bindingantibody, wherein the polypeptide is capable of specifically binding atleast two Spa IgG binding domains A, B, C, D and E of a Staphylococcalprotein A (SpA) polypeptide variant that lacks non-specific Ig-bindingactivity.
 33. The polypeptide of claim 32, wherein the SpA polypeptidethat lacks non-specific Ig-binding activity is SpA_(KKAA).
 34. Thepolypeptide of claim 32 or 33, wherein the polypeptide competes forbinding of SpA_(KKAA) polypeptide with the 5A10, 8E2, 3A6, 7E2, 3F6,1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5,2B8 or 1H7 monoclonal antibody.
 35. The polypeptide of any of claims32-34, wherein the polypeptide has an association constant for theSpA_(KKAA) polypeptide of between about 0.5 and 100×10⁹ M⁻¹, 1.0 and100×10⁹ M⁻¹, or 2.0 and 100×10⁹ M⁻¹ as measured by ELISA.
 36. Thepolypeptide of any of claims 32-35, wherein the polypeptide is a singledomain antibody.
 37. The polypeptide of any of claims 32-36, wherein thepolypeptide is a humanized monoclonal antibody.
 38. The polypeptide ofany of claims 32-37, wherein the purified polypeptide is a humanantibody.
 39. The polypeptide of any of claims 32-37, wherein thepolypeptide is recombinant.
 40. The polypeptide of claim 39, wherein thepurified polypeptide comprises an amino acid region with at least 40%identity to one or more CDR domains from the 5A10, 8E2, 3A6, 7E2, 3F6,F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5,2B8 or 1H7 monoclonal antibodies.
 41. The polypeptide of claim 40,wherein the purified polypeptide comprises two or more amino acidregions that are at least 40% identical to two CDR domains from the5A10, 8E2, 3A6, 7E2, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4,7D11, 2C3, 4C5, 6B2, 4D5, 2B8 or 1H7 monoclonal antibodies.
 42. Thepolypeptide of claim 40, wherein the purified polypeptide comprisesthree amino acid regions that are at least 40% identical to three CDRdomains from the VH or VL domain of the 5A10, 8E2, 3A6, 7E2, 3F6, 1F10,6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5, 2B8 or1H7 monoclonal antibodies.
 43. The polypeptide of any of claims 32-42,wherein the purified polypeptide comprises a sequence at least 40%identical to VH or VL domain of the 5A10, 8E2, 3A6, 7E2, 3F6, 1F10,6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5, 2B8 or1H7 monoclonal antibodies.
 44. The polypeptide of claim 42, wherein thepurified polypeptide comprises six amino acid regions that are at least40% identical to six CDR domains from the VH and VL domains of the 5A10,8E2, 3A6, 7E2, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4, 7D11,2C3, 4C5, 6B2, 4D5, 2B8 or 1H7 monoclonal antibodies.
 45. Thepolypeptide of claim 44, wherein the purified polypeptide comprises theVH domain of the 5A10, 8E2, 3A6, 7E2, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10,4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5, 2B8 or 1H7 monoclonal antibody.46. The polypeptide of claim 44, wherein the purified polypeptidecomprises the VL domain of the 5A10, 8E2, 3A6, 7E2, 3F6, 1F10, 6D11,3D11, 5A11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5, 2B8 or 1H7monoclonal antibody.
 47. The polypeptide of any of claims 32-46, whereinthe recombinant polypeptide comprises one or more CDR domain from aSpA-binding antibody and a scaffold from a polypeptide selected from thegroup consisting of an immunoglobulin, a fibronectin, a lipocalin or aS. aureus protein Z.
 48. The polypeptide of any of claims 32-47, whereinthe purified polypeptide is operatively coupled to a second recombinantpolypeptide that specifically binds to a second Staphylococcal protein.49. The polypeptide of any of claims 32-48, wherein the polypeptide is a5A10, 8E2, 3A6, 7E2, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4,7D11, 2C3, 4C5, 6B2, 4D5, 2B8 or 1H7 monoclonal antibody.
 50. Thepolypeptide of any of claims 32-49, wherein the polypeptide is anantibody comprising (a) a heavy chain comprising said VH region, and ahuman hinge, CH1, CH2, and CH3 regions from an IgG1, IgG2, IgG3 or IgG4subtype; and (b) a light chain comprising said VL region, and either ahuman kappa CL or human lambda CL.
 51. A pharmaceutical compositioncomprising the purified polypeptide of any of claims 32-50.
 52. Thepharmaceutical composition of claim 51, comprising a single unit dose ofthe purified polypeptide in a sealed container.
 53. The pharmaceuticalcomposition of claim 51, comprising at least a second anti-bacterialagent.
 54. The pharmaceutical composition of claim 53, wherein thesecond anti-bacterial agent is a an antibiotic, a Staphylococcal vaccinecomposition or a polypeptide that specifically binds to a secondStaphylococcal protein.
 55. A purified polypeptide that specificallybinds to a Spa variant polypeptide lacking specific Ig-binding activity,wherein the polypeptide has an association constant of 0.5×10⁹ M⁻¹ orgreater for at least two and up to five Spa IgG binding domainsA_(KKAA), B_(KKAA), C_(KKAA), D_(KKAA) and E_(KKAA).
 56. The purifiedpolypeptide of claim 55, wherein the polypeptide comprises an amino acidregion with at least 40% identity to one or more CDR domains from the5A10, 8E2, 3A6, 7E2, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4,7D11, 2C3, 4C5, 6B2, 4D5, 2B8 or 1H7 monoclonal antibodies.
 57. Thepolypeptide of claim 55 or 56, wherein the polypeptide competes forbinding of SpA_(KKAA) polypeptide with the 5A10, 8E2, 3A6, 7E2, 3F6,1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5,2B8 or 1H7 monoclonal antibody.
 58. The polypeptide of any of claims55-57, wherein the polypeptide has an association constant for theSpA_(KKAA) polypeptide of between about 0.5 and 100×10⁹ M⁻¹, 1.0 and100×10⁹M⁻¹, or 2.0 and 100×10⁹ M⁻¹ as measured by ELISA.
 59. Thepolypeptide of any of claims 55-58, wherein the polypeptide is a singledomain antibody.
 60. The polypeptide of any of claims 55-59, wherein thepolypeptide is a humanized monoclonal antibody.
 61. The polypeptide ofany of claims 55-60, wherein the purified polypeptide is a humanantibody.
 62. The polypeptide of any of claims 55-60, wherein thepolypeptide is recombinant.
 63. The polypeptide of claim 62, wherein thepurified polypeptide comprises an amino acid region with at least 40%identity to one or more CDR domains from the 5A10, 8E2, 3A6, 7E2, 3F6,1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5,2B8 or 1H7 monoclonal antibodies.
 64. The polypeptide of claim 63,wherein the purified polypeptide comprises two or more amino acidregions that are at least 40% identical to two CDR domains from the5A10, 8E2, 3A6, 7E2, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4,7D11, 2C3, 4C5, 6B2, 4D5, 2B8 or 1H7 monoclonal antibodies.
 65. Thepolypeptide of claim 63, wherein the purified polypeptide comprisesthree amino acid regions that are at least 40% identical to three CDRdomains from the VH or VL domain of the 5A10, 8E2, 3A6, 7E2, 3F6, 1F10,6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5, 2B8 or1H7 monoclonal antibodies.
 66. The polypeptide of any of claims 55-65,wherein the purified polypeptide comprises a sequence at least 40%identical to VH or VL domain of the 5A10, 8E2, 3A6, 7E2, 3F6, 1F10,6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5, 2B8 or1H7 monoclonal antibodies.
 67. The polypeptide of claim 65, wherein thepurified polypeptide comprises six amino acid regions that are at least40% identical to six CDR domains from the VH and VL domains of the 5A10,8E2, 3A6, 7E2, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4, 7D11,2C3, 4C5, 6B2, 4D5, 2B8 or 1H7 monoclonal antibodies.
 68. Thepolypeptide of claim 67, wherein the purified polypeptide comprises theVH domain of the 5A10, 8E2, 3A6, 7E2, 3F6, 1F10, 6D11, 3D11, 5A11, 1B10,4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5, 2B8 or 11-17 monoclonalantibody.
 69. The polypeptide of claim 67, wherein the purifiedpolypeptide comprises the VL domain of the 5A10, 8E2, 3A6, 7E2, 3F6,1F10, 6D11, 3D11, 5A11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5,2B8 or 1H7 monoclonal antibody.
 70. The polypeptide of any of claims55-69, wherein the recombinant polypeptide comprises one or more CDRdomain from a SpA-binding antibody and a scaffold from a polypeptideselected from the group consisting of an immunoglobulin, a fibronectin,a lipocalin or a S. aureus protein Z.
 71. The polypeptide of any ofclaims 55-70, wherein the purified polypeptide is operatively coupled toa second recombinant polypeptide that specifically binds to a secondStaphylococcal protein.
 72. The polypeptide of any of claims 55-71,wherein the polypeptide is a 5A10, 8E2, 3A6, 7E2, 3F6, 1F10, 6D11, 3D11,5A11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5, 2B8 or 1H7monoclonal antibody.
 73. The polypeptide of any of claims 55-72, whereinthe polypeptide is an antibody comprising (a) a heavy chain comprisingsaid VH region, and a human hinge, CH1, CH2, and CH3 regions from anIgG1, IgG2, IgG3 or IgG4 subtype; and (b) a light chain comprising saidVL region, and either a human kappa CL or human lambda CL.
 74. Apharmaceutical composition comprising the purified polypeptide of any ofclaims 55-73.
 75. The pharmaceutical composition of claim 74, comprisinga single unit dose of the purified polypeptide in a sealed container.76. The pharmaceutical composition of claim 74, comprising at least asecond anti-bacterial agent.
 77. A method of making a neutralizingtherapeutic SpA antibody comprising: a) generating a monoclonal antibodyusing as an antigen a SpA variant that Ig-binding activity; b) screeningmonoclonal antibodies for specific binding to the SpA variant; c)humanizing one or more monoclonal antibodies screened for specificbinding to the SpA variant; and d) screening the one or more humanizedmonoclonal antibodies for an ability to neutralize SpA antibody.